Labelling and selection of molecules

ABSTRACT

A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyze binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting.

[0001] The present invention relates to labelling and selection ofmolecules, such as members of a specific binding pair (sbp) able to binda complementary sbp member of interest, especially though notexclusively a complementary sbp member for which an existing ligand isavailable. In exemplary embodiments, the present invention relates toselection of antibodies, or polypeptides comprising an antibody antigenbinding domain, specific for an antigen of interest for which anexisting binding molecule, which may be an antibody, such as amonoclonal antibody, is already available. It involves deposition of alabel or reporter molecule, such as biotin-tyramine, on molecules in thevicinity of a “marker ligand” which comprises for example a monoclonalantibody (specific for an antigen of interest) in association with anenzyme which catalyzes such deposition. Molecules labelled in accordancewith the present invention may include binding members such asantibodies which bind the same binding target (e.g. antigen) as themarker ligand if such binding members are included in the reactionmedium, the target molecule to which the marker ligand binds, whichallows for identification and/or purification of unknown antigentargets, and/or other molecules in the vicinity of the binding targetand/or the marker ligand when bound to its binding target, e.g. on acell surface on which the binding target is found, including moleculescomplexed with the binding target, allowing for identification of novelprotein-protein interactions. There are also various advantages inlabelling cells or other particles using the present invention,especially when the process is reiterated to augment the extent oflabelling. Further aspects and embodiments of the invention aredisclosed herein.

[0002] Numerous kinds of specific binding pairs are known, as epitomisedby the pair consisting of antibody and antigen. Other specific bindingpairs are discussed briefly infra and may equally be employed in thevarious aspects of the present invention disclosed herein. Forconvenience, however, most of the discussion herein refers to “antibody” as the type of (first) specific binding pair (sbp) member whoseselection is sought in performance of methods of various embodiments ofthe invention, “antigen” as the complementary (second) sbp member ofinterest for which specific binding molecules may be sought to beselected and marker ligand as the pre-existing binding molecule known tobe able to bind the complementary sbp member of interest. Generally, themarker ligand comprises an antibody antigen binding domain specific forthe complementary sbp member of interest (e.g. antigen). Other suitablemarker ligands include hormones, cytokines, growth factors,neuropeptides chemokines, enzyme substrates and any other specificbinding molecule. Also present is a label or reporter molecule and anenzyme that catalyses binding of the label to other molecules in thevicinity.

[0003] Bearing this in mind, the present invention (in some embodiments)can be said to have resulted from the inventors having identified ameans to select for antibodies binding to an antigen, e.g. on cellsurfaces, other solid supports, or in solution, using a marker ligandfor the antigen to guide the recovery of antibodies binding in proximityto the marker ligand. This provides means to label molecules which bindin close proximity to a given defined ligand by transfer of a reportermolecule or label to the binding molecules. The defined ligand occupiesa specific epitope on the antigen and generally blocks that particularepitope, and epitopes overlapping it, from binding other antibodies.Thus, antibodies which are selected for are usually those which do notbind to the marker ligand epitope, but are those which bind neighbouringepitopes. Antibodies which bind the same epitope as the original markerligand may be obtained by an iterative process—using an antibodyobtained in one round of the process as a second marker ligand in afurther round—or by using appropriate conditions, as discussed furtherbelow.

[0004] Signal transfer selection may be used to generate antibodieswhich bind to the same epitope as the marker ligand by re-iterating theselection procedure. Antibodies selected from the first round of signaltransfer selection may be used as new marker ligands for a subsequentround of selection which is carried out in the absence of the originalmarker ligand. This may be referred to as a step-back selection and maybe used to select for antibodies which inhibit the original ligandbinding. If the second stage of a step-back selection is carried out inthe presence of the original marker ligand antibodies which bind themarker ligand-receptor complex, but not the receptor alone, may beselected. Such antibodies may be ligand agonists or antagonists. Ofcourse, step back selection need not be limited to selection fromantibody libraries; any pair of specific binding members can be used insuch a procedure.

[0005] Antibodies which bind epitopes which are nearest to that bound bythe marker ligand have the highest probability of becoming labelled, andthe probability of labelling decreases with distance from the markerligand epitope. Advantageously, the present invention may expedite thepurification of such labelled molecules.

[0006] Transfer of the biotin tyramine reporter molecule may occurwithin up to about 25 nm according to experimental results infra. Thedistance from the binding site of the original marker ligand may beincreased by iteration of the signal transfer process, or by adaptingthe guide molecule by the addition of a spacer between the guidemolecule and the enzyme which catalyses the signal transfer. Such aspacer may be a chemical linker, polymer, peptide, polypeptide, rigidbead, phage molecule, or other particle.

[0007] Such a spacer may be of any suitable desired length, includingabout 10-20 nm, about 20-40 nm, about 40-60 nm, about 60-100 nm, about100 nm or more, such as about 500 nm or more up to about 1 μm or more.

[0008] Furthermore, the labelling and subsequent purification of bindingmolecules specific for antigen of interest which are displayed on thesurface of bacteriophage or other biological particles (see e.g.WO92/01047) facilitates recovery of nucleic acid encoding the specificbinding molecules. In so-called “phage display”, a binding molecule,e.g. antibody or antibody fragment, peptide or polypeptide, e.g. enzyme,is displayed on the surface of a virus particle which contains nucleicacid encoding the displayed molecule. Following selection of particlesthat display molecules with the desired binding specificity, the nucleicacid may be recovered from the particles and used to express thespecific binding molecules or derivatives thereof, which may then beused as desired.

[0009] Other display systems may be used instead of display onfilamentous bacteriophage. Such systems include display on wholebacterial cells or modified bacterial surface structures (Osuna et al.Crit. Rev. Microbiol., 1994, 20: 107-116; Lu et al., BioTechnology,1995, 13: 366-372) and eukaryotic viruses (Boublik et al. BioTechnology,1995, 13: 1079-1084; Sugiyama et al., FEBS Lett., 1995, L 359: 247-250).Bacteriophage display libraries may be generated using fusion proteinswith the gene III protein (e.g. Vaughan et al. Nature Biotechnology,1996, 14: 309-314), or the major gene VIII coat protein (Clackson andWells, Trends Biotechnol., 1994, 12: 173-184), or the gene VI protein(Jespers et al., BioTechnology, 1995, 13: 378-382).

[0010] Herein it is shown that antibodies binding specifically to agiven target antigen, e.g. expressed on the surface of cells, may beselected from a large, diverse phage display library using an existingligand of the desired antigen to guide the selection. It is alsodemonstrated that the desired antigen can be purified from the cells bychemical modification of the antigen in a reaction catalysed by theexisting ligand. Antibodies to any antigen for which a known ligandexists may be obtained in this way, as may antibodies which bindspecifically to the antigen-ligand complex rather than the antigenalone. In addition existing ligands to unknown molecules (e.g. antigens)may be used as markers to guide selection of antibodies to the unknownmolecule or purification of the unknown molecule itself. Surfaceaccessible regions of an antigen may be identified by means of theiraccessibility to labelling, e.g. biotinylation. Biotinylated moleculesmay be cleaved, e.g. proteolytically if they are peptidyl in nature, andbiotinylated fractions detected, e.g. following size fractionation.Furthermore, is the labelling of other molecules in the vicinity of themolecule to which the marker ligand binds allows for those othermolecules to be identified and/or purified for further study. It alsoallows for particular moieties on which the binding target appears to beidentified and/or purified, for instance one cell type displaying aparticular antigen from among a complex mix of different cell types.Determination of the extent of labelling which occurs in the vicinity ofa the molecule to which the marker ligand binds may be used to determinethe copy number of that molecule, e.g. on a cell surface.

[0011] Selection of molecules in accordance with the present inventionis not limited to antigens on cell surfaces. For example, complexproteins with multiple domains or subunits may be coated onto a solidsupport and ligands specific for a particular domain or subunit may beused as marker ligands to guide selection of antibodies to otherneighbouring domains or subunits. A domain or subunit may be conjugated,directly or indirectly, to the enzyme (e.g. HRP) and domain-domain orsubunit-subunit interactions used to guide selection. This may be termed“domain walking”. Marker ligands specific for particular epitopes on aprotein may also be used to guide the selection away from the markerligand epitope and to select for binding molecules which bind otherepitopes within the radius of labelling (e.g. about 25 nm forbiotinylation). This may be termed epitope walking, and example of whichis given in Example 8. A “step-back” selection may be carried out (asdiscussed elsewhere herein), generating a sbp member with the same oroverlapping epitope specificity as the original marker ligand.

[0012] Techniques of the present invention for selection of molecules,which may be known as “signal transfer selection”, need not be limitedto antibody selection; selection from peptide libraries (e.g displayedon phage) may be used to identify peptides with specific bindingcharacteristics for a given protein, which may be any binding domain ortype of ligand interaction, not just antibody/epitope. Example 14illustrates this using peptide libraries to epitope map an antibody(conjugated to HRP) in solution. Libraries or diverse populations ofproteins other than antibodies may be displayed on the surface of phageto allow isolation of novel proteins which bind to a protein inproximity to the marker ligand.

[0013] Signal transfer selection may also be used to chemically modify aparticular cell type possessing a specific antigen to facilitatepurification of that cell type from a background of other cells. Signaltransfer selection may also be applied to the humanisation of existingmonoclonal antibodies since Mab's which recognise an undefined antigenmay be used to target selection of human antibodies with a similarbinding capacity. This may involve the marker ligand including thebinding domain of a non-human antibody, such as a mouse monoclonalantibody, which may be conjugated directly or indirectly to an enzymesuch as HRP. Signal transfer selection may be used to obtain antibodiesfrom a human antibody library displayed on the surface of a suitablevirus, such as bacteriophage or retrovirus, or other biologicalparticle, which bind to the same antigen as the pre-existing non-humanantibody. Repeating the process (“step-back”) using an antibody obtainedin a first performance of the process as the marker ligand in a furtherperformance of the process may be used to obtain human antibodies whichbind to the same epitope as the original non-human antibody—a humanisedantibody. Ability of two binding molecules such as antibodies to bindthe same epitope may of course be assessed using an appropriatecompetition assay.

[0014] Signal transfer selection may be used to generate two antibodies,or other binding members, which bind adjacent epitopes on the sametarget molecule. This provides the potential to generate bispecificantibodies (such as “diabodies”) which may have higher affinities orother desirable biological properties (e.g. neutralising ability) whichthe individual antibodies alone do not exhibit. Signal transferselection may also be used with enzyme substrates to direct selection ofantibodies which bind enzyme active sites and which may be enzymeinhibitors or activators. Direct biotinylation of the enzyme active siteby the substrate may provide a tool to map amino acid residues importantin catalysis.

[0015] A local supply of hydrogen peroxide or other free radical may begenerated by coupling the marker ligand to an enzyme which produces thefree-radical generating enzyme, such as HRP, for example glucoseoxidase, superoxide dismutase or azide. This enables the localgeneration of radicalised biotin-tyramine or other label molecule in thevicinity of the free-radical generating enzyme. An active form offree-radical generating enzyme may be generated in response to a bindingevent, such as the bringing together of two subunits of the enzyme toproduce an active enzyme, or bringing together an activator of theenzyme with the enzyme itself. Radicalised label molecule such asbiotin-tyramine may be thus generated in response to binding events,which may be between specific cell types, proteins, or other specificbinding members.

TERMINOLOGY

[0016] Specific binding member

[0017] This describes a member of a pair of molecules which have bindingspecificity for one another. The members of specific binding pair may benaturally derived or synthetically produced. One member of the pair ofmolecules has an area on its surface, or a cavity, which specificallybinds to and is therefore complementary to a particular spatial andpolar organisation of the other member of the pair of molecules. Thusthe members of the pair have the property of binding specifically toeach other.

[0018] Examples of types of specific binding pairs are antigen-antibody,biotin-avidin/streptavidin, hormone-hormone receptor, receptor-ligand,enzyme-substrate.

[0019] Antibody

[0020] This describes an immunoglobulin whether natural or partly orwholly synthetically produced. The term also covers any polypeptide orprotein having a binding domain which is, or is homologous to, anantibody binding domain. These can be derived from natural sources, orthey may be partly or wholly synthetically produced. Examples ofantibodies are the immunoglobulin isotypes and their isotypicsubclasses; fragments which comprise an antigen binding domain such asFab, scFv, Fv, dAb, Fd; and diabodies.

[0021] It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or othercell producing an antibody may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

[0022] As antibodies can be modified in a number of ways, the term“antibody” should be construed as covering any specific binding memberor substance having a binding domain with the required specificity.Thus, this term covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially synthetic. Chimeric molecules comprising an immunoglobulinbinding domain, or equivalent, fused to another polypeptide aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

[0023] It has been shown that fragments of a whole antibody can performthe function of binding antigens. Examples of binding fragments are (i)the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

[0024] Diabodies are multimers of polypeptides, each polypeptidecomprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

[0025] Antigen binding domain

[0026] This describes the part of an antibody which comprises the areawhich specifically binds to and is complementary to part or all of anantigen. Where an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is termed an epitope. Anantibody antigen binding domain may be provided by one or more antibodyvariable domains. Preferably, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

[0027] Specific

[0028] This refers to the situation in which one member of a specificbinding pair will not show any significant binding to molecules otherthan its specific binding partner (e.g. an affinity of about 1000×worse). The term is also applicable where eg an antigen binding domainis specific for a particular epitope which is carried by a number ofantigens, in which case the specific binding member carrying the antigenbinding domain will be able to bind to the various antigens carrying theepitope.

[0029] Functionally equivalent variant form

[0030] This refers to a molecule (the variant) which although havingstructural differences to another molecule (the parent) retains somesignificant homology and also at least some of the biological functionof the parent molecule, e.g. the ability to bind a particular antigen orepitope. Variants may be in the form of fragments, derivatives ormutants. A variant, derivative or mutant may be obtained by modificationof the parent molecule by the addition, deletion, substitution orinsertion of one or more amino acids, or by the linkage of anothermolecule. These changes may be made at the nucleotide or protein level.For example, the encoded polypeptide may be a Fab fragment which is thenlinked to an Fc tail from another source. Alternatively, a marker suchas an enzyme, flourescein, etc, may be linked.

[0031] Marker ligand

[0032] This refers to one member of a specific binding pair able to bindcomplementary sbp member. In embodiments of the present invention, it isused to guide catalysis of label or reporter molecule deposition at andaround its site of binding to the complementary other member of thespecific binding pair.

[0033] According to a first aspect of the present invention there isprovided a method of labelling molecules, the method including

[0034] providing in a common medium:

[0035] a label molecule;

[0036] a ligand (“first marker ligand”) able to bind a second member ofa specific binding pair (sbp); a said second sbp member;

[0037] an enzyme able to catalyse binding of said label molecule toother molecules, said enzyme being associated with said first markerligand;

[0038] causing or allowing binding of-said first marker ligand to saidsecond sbp member; and

[0039] causing or allowing binding of said label molecule to othermolecules in the vicinity of said first marker ligand bound to saidsecond sbp member.

[0040] A first member of a specific binding pair, such as an antibody,may be included, or a diverse population of such first sbp membersincluding one or more which bind the second sbp member. Molecules towhich the label molecule binds may include a sbp member (“first sbpmember”) which binds said second sbp member.

[0041] Molecules to which the label molecule binds may include a sbpmember (“first sbp member”) which binds a molecule in the vicinity ofsaid second sbp member, as discussed further infra.

[0042] In preferred embodiments of the invention the first sbp member isa polypeptide comprising an antibody antigen binding domain, and thesecond, complementary sbp member is antigen. The marker ligand may be apolypeptide comprising an antibody antigen binding domain, such as amonoclonal antibody or cloned scFv, Fab or other antibody fragment.

[0043] In a preferred embodiment of the present invention, the firstmember of the-specific binding pair is included and is labelled bybinding of the label molecule. This allows identification and/orisolation of-target molecules such as antibodies able to bind asubstance of interest, such as antigen. (The term “target molecules” maybe used to refer to molecules the identification of which is the objectof the person skilled in the art operating the invention.) Suchisolation may be facilitated if the label itself is a member of aspecific binding pair. A preferred label exemplified herein is biotin,able specifically to bind avidin and streptavidin. Also exemplified isthe use of light-activatible streptavidin as the label.

[0044] Following binding of a sbp member label such as biotin to atarget sbp member (e.g. antibody), specific binding of the label to itscomplementary sbp member (e.g. streptavidin in the case of biotinlabelling) may be used in isolation of the target sbp member. Forinstance, streptavidin-coated magnetic beads may be added to the mediumor milieu, allowing streptavidin-biotin binding to take place, thenextracted using a magnet. Sbp members labelled with biotin may then berecovered from the beads.

[0045] Other suitable labels include photo-reactive compounds such asN-[N-4-azido-tetraflurobenzoyl)-biocytinyloxy]-succinimide, orphotoreactive crosslinking agents such as sulfor-SANPAH or SAND(sulfosuccinimidyl2-[m-azido-o-nitrobenzamido]-ethyl-1,3-dithiopropionate) in combinationwith streptavidin or biotin. Conveniently, biotin or other label isconjugated to tyramine, whose covalent binding to peptide molecules iscatalysed by oxygen free radicals generated by hydrogen peroxidase inthe presence of hydrogen peroxide. Instead of biotin-tyramine, labellingin performance of the present invention may employ other forms ofmodified tyramine including fluoresceinated tyramine or other freeradical reagents, such as p-hydroxyphenylpropionyl-biocytin andbiotynil-coumarin galactose. Labels such as biotin (e.g. asbiotin-tyramine) may be preferred over photo-reactive labels, e.g.because of ease of handling, though Example 10 below demonstratesoperation of the present invention using a label whose binding islight-activated, i.e. SAND linked to streptavidin. An advantage of usinga light-activatable label, such as streptavidin-SAND, is the distanceover which this label can be deposited. The linker between thestreptavidin and SAND is 1.8 nm so the proximity within which thestreptavidin is deposited is up to a maximum of about 1.8 nm, comparedwith a radius of up to about 25 nm of biotinylation which is obtainablewith biotin-tyramine.

[0046] The enzyme that catalyses binding of the label molecule to othermolecules may be associated with the marker ligand by any suitable meansavailable in the art. It may be conjugated directly, e.g. via a peptidebond (in which case a fusion protein comprising marker ligand and enzymemay be produced by expression from encoding nucleic acid), or bychemical conjugation of the marker ligand and enzyme, or indirectly.Indirect conjugation of enzyme and marker ligand may conveniently beachieved using a further binding molecule that forms a specific bindingpair with the marker ligand. For example, the marker ligand may be amouse monoclonal antibody, or may comprise a mouse antibody sequence,and the enzyme may be provided conjugated to an anti-mouse antibody orantibody antigen binding domain (e.g. as a fusion protein). Binding ofanti-mouse antibody to the mouse monoclonal, itself binding the antigenof interest (second sbp member), brings the conjugated enzyme into closeproximity with the antigen and any molecules in the medium or milieuable to bind the antigen (e.g. target antibodies), allowing the enzymeto catalyse labelling of such molecules (e.g. target antibodies) and/orthe antigen. Labelled molecules may be identified and/or isolated forinvestigation and/or use.

[0047] As mentioned already, the first sbp member when provided in thereaction milieu may be one of a diverse population of that type of sbpmember with different binding specificities. Such a population may beprovided by expression from a genetically diverse repertoire of nucleicacid sequences. In the case of antibody antigen binding domains, thesemay be provided by expression from a repertoire of rearranged orunrearranged immunoglobulin sequences from an organism (preferablyhuman) which has or has not been immunised with the antigen of interest.A repertoire of sequences encoding antibody antigen binding domains (VHand/or VL) may additionally or alternatively be provided by any ofartificial rearrangement of V, J and D gene segments, mutation in vitroor in vivo, in vitro polynucleotide synthesis and/or any other suitabletechnique available in the art. Suggested references include Vaughan etal., (1996) Nature Biotechnology 14: 309-314; Griffiths et al., (1993)EMBO J. 12: 725-734.

[0048] Conveniently, a diverse population of binding molecules isprovided displayed on the surface of a biological particle such as avirus, e.g. bacteriophage, each particle containing nucleic acidencoding the binding molecule displayed on its surface. WO92/01047discloses in detail various formats for “phage display” of polypeptidesand peptide binding molecules, such as antibody molecules, includingscFv, Fab and Fv fragments, and enzymes, both monomeric and polymeric.Following labelling of phage displaying a target sbp member able to bindcomplementary sbp member of interest, and isolation of these from thereaction medium or milieu as discussed, nucleic acid may be recoveredfrom phage particles. This nucleic acid may be sequenced if desired.

[0049] Other display Systems, e.g. on bacterial cells or retroviruses,are applicable, as has been mentioned already.

[0050] The nucleic acid taken from the particle, or its nucleotidesequence, may be used to provide nucleic acid for production of theencoded polypeptide or a fragment or derivative thereof in a suitableexpression system, such as a recombinant host organism. A derivative maydiffer from the starting polypeptide from which it is derived by theaddition, deletion, substitution or insertion of amino acids, or by thelinkage of other molecules to the encoded polypeptide. These changes maybe made at the nucleotide or protein level. For example the encodedpolypeptide may be a Fab fragment which is then linked to an Fc tailfrom another source. Alternatively markers such as enzymes, flouresceinsetc may be linked to eg Fab, scFv fragments.

[0051] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells and many others. A common, preferred bacterial host is E. coli.

[0052] Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may beplasmids, viral e.g. ′phage, or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Short Protocols inMolecular Biology, Second Edition, Ausubel et al. eds., -John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

[0053] The expression end product may be used to prepare a compositioncomprising the expression end product or a derivative thereof andoptionally one or more further components such as a pharmaceuticallyacceptable vehicle, carrier or excipient, which may for example be usedas a therapeutic or prophylactic medicament or a diagnostic product.

[0054] In some embodiments of the present invention, the second sbpmember (to which the marker ligand binds —e.g. antigen) is labelled.This is useful if the target molecule is an unknown antigen/receptor forthe known marker ligand (e.g. monoclonal antibody or the natural ligandfor the antigen/receptor). In such case, the first sbp member may beomitted from the reaction medium or milieu. Following labelling of thesecond sbp member it may be identified and/or isolated in accordancewith procedures disclosed herein.

[0055] According to a further aspect of the present invention there isprovided reaction medium or milieu containing:

[0056] a member of said specific binding pair;

[0057] a label molecule;

[0058] a ligand (“marker ligand”) able to bind said sbp member;

[0059] an enzyme able to catalyse binding of said label molecule toother molecules, said enzyme being associated with said marker ligand;

[0060] as provided in methods according to the invention. A further sbpmember (designated “first”) may be present, in which case the markerligand is able to bind complementary second sbp member.

[0061] A further aspect of the present invention provides a sbp memberidentified as having ability to bind complementary sbp member ofinterest and/or isolated using a method as disclosed herein, including areceptor or ligand identified and/or isolated as disclosed, andcompositions comprising such an identified and/or isolated sbp memberand nucleic acid encoding the identified and/or isolated sbp member.

[0062] The present invention generally provides for any specific bindingmember identified by virtue of its ability to bind to complementary sbpmember in close proximity (e.g. less than about 25 nm, and possibly lessthan about 20 nm, less than about 15 nm, less than about 10 nm, about5-10 nm or about 5 nm) to an existing defined ligand, which may betermed a “marker ligand” and is used to guide catalysis of reportermolecule deposition on to the specific binding member.

[0063] The invention also provides for the use of the methods and meansprovided herein for the selection of phage-displayed sbp members, e.g.antibodies, peptides or proteins, also the selection or identificationof unknown receptors using a known ligand, either by directed labellingof the receptor, or by production of an antibody against the receptor,followed by immuno-purification.

[0064] The invention also provides for the use of signal transferselection in an iterative manner, i.e. using one or more sbp membersselected in a cycle to select for further sbp members. This may be usedto select sbp members which are capable of acting as antagonists oragonists to the original marker ligand used in the first stage of theselection.

[0065] Cell-surface or other receptors may be identified in a processaccording to the present invention by conjugating a ligand for theuncharacterised receptor (e.g. the natural of the receptor) with anenzyme able to catalyse binding of the label molecule. Binding of theligand to the receptor, e.g. on cells expressing it, may then be carriedout in the presence or absence of sbp members, such as antibodies,particularly a library of sbp members, e.g. displayed on phage, and thelabel molecule. The natural ligand may transfer the signal moleculedirectly onto the unknown receptor. Labelled receptor may then bedirectly purified, e.g. from a cell extract, and may be proteinsequenced. In the presence of the sbp members, e.g. a library ofantibodies displayed on phage, signal transfer will generate labelledsbp members which are able to bind the receptor. These may then be usedto generate purified receptor by affinity purification.

[0066] The invention also provides for the use of such processes toidentify unknown ligands for known receptors, either by directedlabelling of the ligand, or by production of an antibody directedagainst the ligand followed by immuno-purification.

[0067] Further provided by the invention is the use of signal transferselection to guide the selection of antibodies to a given epitope,domain or subunit of a protein or complex by an existing ligand orantibody which recognises a neighbouring epitope, domain or subunit.Existing sbp's (e.g. monoclonal antibodies) to a defined but perhapsundesirable epitope, subunit or region of a protein complex may beconjugated to an enzyme capable of catalysing binding of the labelmolecule to other molecules. These conjugated sbp's may then be used todirect signal transfer of the label to other sbp members, e.g.antibodies (e.g. on phage), binding to the same antigen but atnon-identical, non-overlapping, but neighbouring epitopes which may beon adjacent subunits of a protein, or on adjacent regions of a proteincomplex.

[0068] Signal transfer selection may be used to obtain antibodies orother binding molecules which bind to the same epitope as the markerligand. For example, sub-saturating amounts of the marker ligand may beadded to a mutlimeric protein and the marker ligand may then directselection of binding specificities recognising the same epitope as themarker ligand, but on a neighbouring subunit, or copy of the multimer.The marker ligand may be capable of labelling binding species which bindto the same epitope if labelling occurs concomitantly with the markerligand being competed off the target protein by the species which isbeing selected for.

[0069] Another application of the process is that of selecting forantibodies or other ligands which bind to a particular cell structure orcell type.

[0070] Further aspects of the present invention arise from the genecloning work described in Example 16. Encoding nucleic acid, isolatedpolypeptides, specific binding molecules for the polypeptide and othermolecules which interact with the polypeptide, particularly those whichmodulate its function, e.g. interfere with its association with CC-CKR5and/or other polypeptide in the vicinity of CC-CKR5 on the surface ofCD4+ cells, other molecules which interact with the polypeptide, andmethods and uses of these are all provided by the present invention.

[0071] Nucleic acid according to this aspect of the present inventionmay include or consist essentially of a nucleotide sequence encoding apolypeptide which includes an amino acid sequence shown in FIG. 8.

[0072] The coding sequence may be that shown in FIG. 8, or it may be amutant, variant, derivative or allele of the sequence shown. Thesequence may differ from that shown by a change which is one or more ofaddition, insertion, deletion and/or substitution of one or morenucleotides of the sequence shown. Changes to a nucleotide sequence mayresult in an amino acid change at the protein level, or not, asdetermined by the genetic code.

[0073] Thus, nucleic acid according to the present invention may includea sequence different from the sequence shown in FIG. 8 yet encode apolypeptide with-the same amino acid sequence. The polypeptide mayinclude a sequence of about 60 contiguous amino acids from FIG. 8, morepreferably about 70 contiguous amino acids, more preferably about 80. Anamino acid sequence from the second reading frame may be preferred. Astop codon occurs in this frame at nucleotide 251, so in a preferredembodiment the polypeptide includes a contiguous sequence of amino acidsencoded by the nucleotide sequence of the second reading frame of FIG. 8up to said stop codon. Usually, additional amino acids are includedN-terminal to the amino acid sequence shown.

[0074] On the other hand, the encoded polypeptide may include an aminoacid sequence which differs by one or more amino acid residues from therelevant amino acid sequence shown in FIG. 8. Nucleic acid encoding apolypeptide which is an amino acid sequence mutant, variant, derivativeor allele of a sequence shown in FIG. 8 is further provided by thepresent invention.

[0075] Nucleic acid encoding such a polypeptide may show at thenucleotide sequence and/or encoded amino acid level greater than about50% homology with the relevant coding/amino acid sequence shown in FIG.8, greater than about 60% homology, greater than about 70% homology,greater than about 80% homology, greater than about 90% homology orgreater than about 95% homology.

[0076] As is well-understood, homology at the amino acid level isgenerally in terms of amino acid similarity or identity. Similarityallows for “conservative variation”, such as substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Similarity may be as defined and determined by the TBLASTNprogram, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which isin standard use in the art. Homology may be over the full-length of therelevant amino acid sequence of FIG. 8, or may more preferably be over acontiguous sequence of about 20, 25, 30, 40, 50, 60, 70, 80 or moreamino acids, compared with the relevant amino acid sequence of FIG. 8.

[0077] At the nucleic acid level, homology may be over the full-lengthor more preferably by comparison with the a contiguous nucleotide codingsequence within the sequence of FIG. 8 of about 50, 60, 70, 80, 90, 100,120, 150, 180, 210, 240 or more nucleotides.

[0078] Generally, nucleic acid according to the present invention isprovided as an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. Where nucleic acid according to theinvention includes RNA, reference to the sequence shown should beconstrued as reference to the RNA equivalent, with U substituted for T.

[0079] Nucleic acid sequences encoding all or part of the gene and/orits regulatory elements can be readily prepared by the skilled personusing the information and references contained herein and techniquesknown in the art (for example, see Sambrook, Fritsch and Maniatis,“Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, and Ausubel et al, Short Protocols in Molecular Biology,John Wiley and Sons, 1992).

[0080] The sequence information provided in FIG. 8 enables cloning ofthe full-length human coding sequence. The present invention provides amethod of obtaining nucleic acid of interest, the method includinghybridisation of a probe having the sequence shown in FIG. 8 or acomplementary sequence, or a suitable fragment of either, to targetnucleic acid. Hybridisation is generally followed by identification ofsuccessful hybridisation and isolation of nucleic acid which hashybridised to the probe, which may involve one or more steps of PCR. Thenucleic acid sequences provided herein readily allow the skilled personto design PCR primers for amplification of the full-length sequence.

[0081] Nucleic acid according to the present invention is obtainableusing one or more oligonucleotide probes or primers designed tohybridise with one or more fragments of the nucleic acid sequence shownin FIG. 8 particularly fragments of relatively rare sequence, based oncodon usage or statistical analysis. A primer designed to hybridise witha fragment of the nucleic acid sequence shown in FIG. 8 may be used inconjunction with one or more oligonucleotides designed to hybridise to asequence in a cloning vector within which target nucleic acid has beencloned, or in so-called “RACE” (rapid amplification of cDNA ends) inwhich cDNA's in a library are ligated to an oligonucleotide linker andPCR is performed using a primer which hybridises with the sequence shownin FIG. 8 and a primer which hybridises to the oligonucleotide linker.

[0082] Such oligonucleotide probes or primers, as well as thefull-length sequence (and mutants, alleles, variants and derivatives)are also useful in screening a test sample containing nucleic acid forthe presence of alleles, mutants and variants, with diagnostic and/orprognostic implications.

[0083] Nucleic acid isolated and/or purified from one or more cells(e.g. human) or a nucleic acid library derived from nucleic acidisolated and/or purified from cells (e.g. a cDNA library derived frommRNA isolated from the cells), may be probed under conditions forselective hybridisation and/or subjected to a specific nucleic acidamplification reaction such as the polymerase chain reaction (PCR), asdiscussed.

[0084] In the context of cloning, it may be necessary for one or moregene fragments to be ligated to generate a full-length coding sequence.Also, where a full-length encoding nucleic acid molecule has not beenobtained, a smaller molecule representing part of the full molecule, maybe used to obtain full-length clones. Inserts may be prepared frompartial cDNA clones and used to screen cDNA libraries. The full-lengthclones isolated may be subcloned into expression vectors and activityassayed by transfection into suitable host cells, e.g. with a reporterplasmid.

[0085] Those skilled in the art are well able to employ suitableconditions of the desired stringency for selective hybridisation, takinginto account factors such as oligonucleotide length and basecomposition, temperature and so on.

[0086] On the basis of amino acid sequence information, oligonucleotideprobes or primers may be designed, taking into account the degeneracy ofthe genetic code, and, where appropriate, codon usage of the organismfrom the candidate nucleic acid is derived. An oligonucleotide for usein nucleic acid amplification may have about 10 or fewer codons (e.g. 6,7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or24). Generally specific primers are upwards of 14 nucleotides in length,but not more than 18-20. Those skilled in the art are well versed in thedesign of primers for use processes such as PCR. Various techniques forsynthesizing oligonucleotide primers are well known in the art,including phosphotriester and phosphodiester synthesis methods.

[0087] A further aspect of the present invention provides anoligonucleotide or polynucleotide fragment of the nucleotide sequenceshown in FIG. 8, or a complementary sequence, in particular for use in amethod of obtaining and/or screening nucleic acid. Somepreferred-oligonucleotides have a sequence shown in FIG. 8 or a sequencewhich differs from any of the sequences shown by addition, substitution,insertion or deletion of one or more nucleotides, but preferably withoutabolition of ability to hybridise selectively with nucleic acid with thesequence shown in FIG. 8, that is wherein the degree of homology of theoligonucleotide or polynucleotide with one of the sequences given issufficiently high.

[0088] Nucleic acid according to the present invention may be used inmethods of gene therapy, for instance in treatment of individuals withthe aim of preventing or curing (wholly or partially) a disease. Thismay ease one or more symptoms of the disease.

[0089] A convenient way of producing a polypeptide according to thepresent invention is to express nucleic acid encoding it, by use of thenucleic acid in an expression system.

[0090] Accordingly, the present invention also encompasses a method ofmaking a polypeptide (as disclosed), the method including expressionfrom nucleic acid encoding the polypeptide (generally nucleic acidaccording to the invention). This may conveniently be achieved bygrowing a host cell in culture, containing such a vector, underappropriate conditions which cause or allow expression of thepolypeptide. Polypeptides may also be expressed in in vitro systems,such as reticulocyte lysate.

[0091] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

[0092] Nucleic acid may be introduced into a host cell and this may befollowed by causing or allowing expression from the nucleic acid, e.g.by culturing host cells (which may include cells actually transformedalthough more likely the cells will be descendants of the transformedcells) under conditions for expression of the gene, so that the encodedpolypeptide is produced. If the polypeptide is expressed coupled to anappropriate signal leader peptide it may be secreted from the cell intothe culture medium. Following production by expression, a polypeptidemay be isolated and/or purified from the host cell and/or culturemedium, as the case may be, and subsequently used as desired, e.g. inthe formulation of a composition which may include one or moreadditional components, such as a pharmaceutical composition whichincludes one or more pharmaceutically acceptable excipients, vehicles orcarriers (e.g. see below).

[0093] The skilled person can use the techniques described herein andothers well known in the art (for which see e.g. the Sambrook andAusubel references cited herein) to produce large amounts ofpolypeptide, or fragments or active portions thereof, for use aspharmaceuticals, in the developments of drugs and for further study intoits properties and role in vivo.

[0094] Thus, a further aspect of the present invention provides apolypeptide which includes an amino acid sequence shown in FIG. 8 asdiscussed, which may be in isolated and/or purified form, free orsubstantially free of material with which it is naturally associated,such as other polypeptides or such as human polypeptides other thanpolypeptide or (for example if produced by expression in a prokaryoticcell) lacking in native glycosylation, e.g. unglycosylated.

[0095] Polypeptides which are amino acid sequence variants, alleles,derivatives or mutants are also provided by the present invention, ashas been discussed. Preferred such polypeptides have function, that isto say have one or more of the following properties: immunologicalcross-reactivity with an antibody reactive with a polypeptide for whichthe sequence is given in FIG. 8; sharing an epitope with a polypeptidefor which the amino acid sequence is shown in FIG. 8 (as determined forexample by immunological cross-reactivity between the two polypeptides.

[0096] The present invention also includes active portions, fragments,derivatives and functional mimetics of the polypeptides of theinvention. A fragment of the polypeptide may be a stretch of amino acidresidues of at least about five to seven contiguous amino acids, oftenat least about seven to nine contiguous amino acids, typically at leastabout nine to 13 contiguous amino acids and, most preferably, at leastabout 20 to 30 or more contiguous amino acids. Fragments of thepolypeptide sequence antigenic determinants or epitopes useful forraising antibodies to a portion of the amino acid sequence.

[0097] A polypeptide, peptide fragment, allele, mutant or variantaccording to the present invention may be used in phage display or othertechnique (e.g. involving immunisation) in obtaining specificantibodies. Antibodies are useful in purification and other manipulationof polypeptides and peptides, diagnostic screening and therapeuticcontexts.

[0098] The provision of the novel polypeptides enables for the firsttime the production of antibodies able to bind it specifically, and byprocedures other than the signal transfer selection which led to itsidentification and the isolation of antibody CD4E1 as described inExample 16. Accordingly, a further aspect of the present inventionprovides an antibody able to bind specifically to a polypeptideincluding a sequence given in FIG. 8.

[0099] Such antibodies may be obtained by selection on peptides orproteins including amino acid sequences of FIG. 8, e.g. using phagedisplay libraries as in WO92/01047, or by using such peptides orproteins to immunise animals and obtain monoclonal antibodies orpolyclonal antisera.

[0100] Antibodies identified, e.g. by phage display, may then be used toidentify further proteins, e.g. receptor molecules, which may becomplexed with the protein including the amino acid sequence of FIG. 8,using techniques of signal transfer selection as disclosed herein.

[0101] cDNA expression libraries, for example displayed on phage, may beused in conjunction with signal transfer selection to identify ligandswhich bind molecules, such as receptors, in the vicinity of proteinincluding the amino acid sequence of FIG. 8. An antibody, e.g. with amyc tag, may bind to the protein on the surface of CD4 lymphocytes. thephage-displayed cDNA expression library may be added, followed by theantibody 9E10 (which binds to the myc tag) conjugated to HRP. Adition ofbiotin-tyramine would then lead to the labelling of molecules in thevicinity of the antibody, including phage expressing receptor ligands.The antibody CD4E1 would be suitable for this.

[0102] The polypeptides, antibodies, peptides and nucleic acid of theinvention may be formulated-in a composition. Such a composition mayinclude, in addition to one of the above substances, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g. oral, intravenous, cutaneousor subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0103] A polypeptide according to the present invention may be used inscreening for molecules which affect or modulate its activity orfunction, including ability to interact or associate with anothermolecule, such as CC-CKR5 or other molecule, e.g. on the surface of CD4+cells. Such molecules may be useful in a therapeutic (possibly includingprophylactic) context.

[0104] A method of screening for a substance which modulates activity ofa polypeptide may include contacting one or more test substances withthe polypeptide in a suitable reaction medium, testing the activity ofthe treated polypeptide and comparing that activity with the activity ofthe polypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

[0105] Combinatorial library technology provides an efficient way oftesting a potentially vast number of different substances for ability tomodulate activity of a polypeptide. Such libraries and their use areknown in the art. The use of peptide or protein libraries may bepreferred.

[0106] As an alternative to using signal transfer selection to identifymolecules which interact with protein including an amino acid sequenceshown in FIG. 8, test substances may be screened for ability to interactwith the polypeptide, e.g. in a two-hybrid system (which requires thatboth the polypeptide and the test substance can be expressed, e.g. in acell such as a yeast or mammalian cell, from encoding nucleic acid).This may be used as a coarse screen prior to testing a substance foractual ability to modulate activity of the polypeptide. The screen maybe used to screen test substances for binding to a specific bindingpartner, to find mimetics of polypeptide, e.g. for testing asanti-tumour therapeutics. Two-hybrid screens may be used to identify asubstance able to modulate, e.g interfere with, interaction between twopolypeptides or peptides.

[0107] The two-hybrid screen assay format is described by Fields andSong, 1989, Nature 340; 245-246. This type of assay format can be usedin both mammalian cells and in yeast. Various combinations of DNAbinding domain and transcriptional activation domain are available inthe art, such as the LexA DNA binding domain and the VP60transcriptional activation domain, and the GAL4 DNA binding domain andthe GAL4 transcriptional activation domain. Suitable fusion constructsare produced for expression within the assay system. When screening fora susbstance able to modulate an interaction between two components,test substances (e.g. in a combinatorial peptide library) may beexpressed from a third construct.

[0108] Following identification of a substance which modulates oraffects polypeptide activity and/or its ability to interact with orassociate with another molecule, the substance may be investigatedfurther. Furthermore, it may be manufactured and/or used in preparation,i.e. manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0109] Thus, the present invention extends in various aspects not onlyto a substance identified using a nucleic acid molecule as a modulatorof polypeptide activity, in accordance with what is disclosed herein,but also a pharmaceutical composition, medicament, drug or othercomposition comprising such a substance, a method comprisingadministration of such a composition to a patient, e.g. for treatment(which may include preventative treatment) of cancer, use of such asubstance in manufacture of a composition for administration, e.g. fortreatment of cancer, and a method of making a pharmaceutical compositioncomprising admixing such a substance with a pharmaceutically acceptableexcipient, vehicle or carrier, and optionally other ingredients.

[0110] Further aspects of the invention and embodiments will be apparentto those skilled in the art. All documents mentioned herein areincorporated by reference. In order that the present invention may befully understood the following examples are provided by way ofexemplification only and not by way of limitation. Reference is made tothe following figures:

[0111]FIG. 1 shows a schematic representation of a process according toone embodiment of the present invention. A target antibody able to bindthe antigen of interest (CEA) is labelled by biotinylation because itbinds the antigen in the region of binding of a marker ligand whichcomprises a monoclonal antibody specific for CEA joined to the enzymehydrogen peroxidase. In the presence of hydrogen peroxide, the hydrogenperoxidase catalyses binding of biotin-tyramine to molecules in thevicinity of the enzyme, including the target antibody(CEA—carcinoembronic antigen; HRP—hydrogen peroxidase;BT—Biotin-tyramine.)

[0112]FIG. 2 illustrates results obtained in experiments described inExample 12, showing the distribution of gold particles at the ends ofpage. For different numbers of beads per phage end the frequency isplotted. The average number of particles per phage was 6.6, the detectedrange 5 nm to 25 nm. The diameter of a globular protein is 4 nm.

[0113]FIG. 3 illustrates a “step-back” selection scheme as exemplifiedexperimentally in Example 13.

[0114]FIG. 3(a) illustrates a process in which HRP-marker ligandconjugate directs the signal transfer of biotin tyramine (ET) onto phagebinding around the ligand. Biotinylated phage are then allowed to bindcells in the absence of ligand, as shown in FIG. 3(b).

[0115]FIG. 3(b) shows binding of biotinylated phage in the absence ofthe original marker ligand. Streptavidin-HRP is added and a new aliquotof phage library then added (illustrated in black) which can then bebiotinylated by signal transfer and selected. In the illustratedembodiment, the selected phage mimics the ligand and inhibits itsbinding to cells.

[0116]FIG. 4 shows results of flow cytometry experiments described inExample 18. The peak position (i.e. a measure of the fluorscenceachieved) obtained using different biotin tyramine concentrations (inμg/ml) is plotted.

[0117]FIG. 5 shows the fluorescence shifts resulting from two flowcytometry readings, one for a sample subject to one biotin tyraminetreatment, the other for a sample subject to reiteration, as describedin-Example 19. As can be seen, iteration of the biotin tyramine treamentresults in a 2.5 fold shift in the average fluorescence level of thecells. (Events plotted against FL1LOG.)

[0118]FIG. 6 shows the results of flow cytometry experiments describedin Example 20. (Events against FL1LOG.)

[0119]FIG. 6(a) shows results with mononuclear cells from blood labelledwith anti-CD36.

[0120]FIG. 6(b) shows results for control enrichment, no CD36 antibodyadded at the start.

[0121]FIG. 6(c) shows results for enriched cells labelled withanti-CD36.

[0122]FIG. 7 shows the results of experiments described in Example 21.Phage recovered (×10⁵) is plotted for various concentrations ofbiotin-tyramine in μg/ml.

[0123]FIG. 8 shows nucleotide and amino acid sequences for the humanhomologue of the rat gene CL-6 identified for the first time in the workdescribed in Example 16. EcoRI cloning sites are underlined.

[0124] For one specific embodiment of the present invention, theprocedure may be summarised as follows, for purposes of illustration.

[0125] The exemplary system is based upon the use of immobilisedreporter enzyme to catalyse the deposition of multiple copies ofbiotinylated tyramine molecules around the site of enzyme activity.Catalysed enzyme reporter deposition (CARD) has been used as a means ofsignal amplification in immunocytochemistry, ELISA and blotting formats(Bobrow et al. (1992) J. Immunol. Methods, 125: 279-285). The inventionhere comes from the realisation that the deposition of a reportermolecule can be used not only as an amplification system, but also as atransfer system which allows recovery of tagged ligands.

[0126] In the example described here, horseradish peroxidase (HRP)activity is used to catalyse biotinylated tyramine molecule deposition.HRP activity is targeted to a specific site of interest, e.g. on a cellsurface, by the use of a primary mouse Mab with a desired bindingspecificity, the HRP activity being provided by an anti-mouse-HRPconjugated second antibody which recognises the primary Mab. HRPactivity may alternatively be provided by direct conjugation of the Mabor ligand to the enzyme (e.g. by expression as a fusion protein). Phageparticles displaying antibody antigen binding domains are incubated onthe cell surface along with the primary Mab, and those binding aroundthe site of the primary Mab, and hence around the site of HRP activity,become covalently linked to biotin tyramine molecules. This reaction iscatalysed by oxygen free radicals generated by the HRP in the presenceof H₂O₂ (FIG. 1).

[0127] Biotinylated phage may then be specifically recovered usingstreptavidin coated magnetic beads and hence phage which bind in closeproximity to the existing mouse Mab are enriched for. The half life ofthe biotin-tyramine phenolic free radical is very short, so depositionoccurs extremely close to the activating enzyme (Bobrow et al., supra).When CARD is used as an amplification system to enhance signal inimmunocytochemistry no detectable loss of image resolution is apparent,indicating that deposition occurs in close association with thecatalytic enzyme (Adams, J. C. (1992) J. Histochem. and Cytol. 40:1457-1463). The area over which the signal transfer occurs may beincreased or decreased by modifying the viscosity or temperature of thesolution in which the reaction is carried out, or by adding excessunbiotinylated tyramine.

[0128] Signal transfer selection has general applications to theidentification of protein-protein interactions and in some ways isanalogous to the yeast two-hybrid system which has proved to be a verypowerful technique for the detection of such interactions (Fields andSong, 1989, Nature 340, 245-246). Both systems involve a tagged knownprotein which can be paired with a library of unknown proteins, some ofwhich may interact with the tagged protein. Interaction between the twoproteins in the two hybrid system results in transcriptional activationof the yeast GAL1-lacZ gene which encodes enzymes for galactoseutilisation and hence allows selection of the interacting clone ongalactose-containing media. Interaction of the two proteins in thesignal transfer system results in labelling of the unknown protein, e.g.phage-displayed antibody, peptide or other protein and hence recovery ofthat moiety. If phage-displayed antibody, peptide or other protein isthe labelled (e.g biotinylated) element then rescue of the gene for theinteracting protein is facilitated, since in phage display each phageparticle contains nucleic acid encoding the antibody, peptide or otherprotein it displays (see e.g. WO92/01047). Signal transfer selection isnot confined to intracellular expression in yeast, and as such has manyadvantages over the yeast two-hybrid system.

[0129] Examples 15 and 16 demosntrate how signal transfer selection maybe used as a tool for discovering novel protein-protein interactions.Examples of the types of protein-protein interactions which may beidentified include proteins interacting in signal transduction pathways,such as G proteins, kinases, phosphatases. Receptors often exist asmultiprotein complexes, interacting pairs of which may be identifiedeither in the presence or absence of ligand binding. Protein-proteininteractions which occur within the cell may also be identified, forinstance using cell extracts, inside-out vesicles, nuclear extracts andextracts from other cellular compartments, either in solution orimmobilised on a solid support. The present invention may also beapplied to the identification of protein-DNA interactions. Segments ofDNA encorporating putative transcription factor binding domains may belabelled (e.g. biotinylated) and coupled to enzyme-associated bindingmolecule for the label (e.g. streptavidin). Proteins which bind the DNAsequence may be selected by signal transfer selection.

[0130] There are many applications of signal transfer selections, whichwill be evident to people skilled in the art. Applications include theisolation of antibodies which specifically recognise a ligand-receptorcomplex, using an enzyme conjugate ligand to target the selection ofsuch antibodies. Specific labelling of one cell type over and abovebackground cell types may be achieved. For example, cells expressing oneparticular surface antigen may be labelled using an enzyme-conjugatedsbp member which recognises that antigen and which can transfer label tothose cells alone. This allows purification of the antigen-expressingcell type from a background of cells which do not express the antigenand do not, therefore, become labelled (or not significantly so). Thisis exemplified in Example 20.

[0131] Signal transfer labelling need not be limited to cell surfaces.Any protein, virus particle or other species in a complex mix may belabelled specifically and purified away from the unlabelled population.

[0132] Signal transfer has applications to signal enhancement in flowcytometry, as discussed and demonstrated in Examples 18 and 19. Thesignal enhancement profile may be used for particular molecules, e.g. ona cell surface, to assess copy number of that molecule, e.g. on aparticular cell or cell type, or to asess the proximity of two or moredifferent target molecules, e.g. on the same cell, as well as providinga more sensitive method for detection of a particular protein, e.g. on acell surface.

[0133] Another application is that of reverse drug screening. In thisprocess a drug which is known to be efficacious, but the cellular targetof which is unknown, may be conjugated to the enzyme which directs labeldeposition. The drug-enzyme complex may then be incubated with cellularextracts and the labelling molecule added. Proteins in the cellularextract which bind to the drug-enzyme conjugate then become labelled,allowing for their purification and characterisation.

[0134] Since the signal transfer selection mechanism relies on thegeneration of free radicals use may be made of the generation of freeradicals by a protein or putative enzyme to select for a protein withnovel or enhanced catalytic activity. Phage libraries of proteins,enzymes, or putative catalytic antibodies may be made and selection maybe directed by the labelling (e.g. biotinylation) of active species dueto their ability to generate free radicals which activate the label(e.g. biotin tyramine) and cause its deposition on the phage displayedspecies.

[0135] Signal transfer technology also has a number of in vivoapplications, for example in tumour targeting. An antibody-HRP conjugatewhich specifically recognises a tumour type may be allowed to localiseto the tumour in vivo. Biotin tyramine, or a similar molecule, may thenbe injected, and the HRP may catalyse biotin tyramine depositionspecifically at the tumour site. This would result in a heavilybiotinylated tumour to which streptavidin-conjuagte drugs, orstreptavidin-liposomes as vechicles for gene therapy or drug delivery,may be targeted.

[0136] Signal transfer is a process which can be re-iterated resultingin the successive build up of biotin tyramine molecules around a focusof enzyme activity. This may have In vivo applications e.g. in thecontext of arteriole or nerve repair since successive layers of biotintyramine, or similar molecules, may be depositied at sites of damage togenerate complexes which may block damaged vessels.

[0137] The iterative potential of biotin-tyramine and other labeldesposition in accordance with the present invention may be used in thegeneration of oriented surfaces. Successive layers of proteins, or otherspecies, may be deposited on the surface. An initial protein, or otherspecies, may be immobilised on a surface and a binding molecule specificfor this initial protein may be enzyme (e.g. HRP-) conjugated andallowed to bind to the surface, then used to deposit a layer of biotinetyramine over the initial surface. A second, e.g., streptavidin-linkedprotein, or other species, may then be added to the surface, giving alayer of the second protein. This process may be re-iterated as requiredto build up complex oriented layers on surfaces.

[0138] A model system has been used to exemplify the potential of thisinvention utilising a HeLa cell line which has been transfected with thegene for human carcinoembryonic antigen (CEA). A scFv which specificallyrecognises CEA has been used for initial experiments and a large scFvphage display library has been used to generate further anti-CEAspecific scFv's using the signal transfer selection system.

[0139] Further experiments have been carried out to select for specificcell surface proteins on cultured human endothelial cells.

[0140] List of examples

[0141] EXAMPLE 1—Recovery of CEA-binding phage from the surface of cellsexpressing CEA in the presence or absence of a marker anti-CEA mouseantibody.

[0142] EXAMPLE 2—Selection of human CEA-binding phage from a largelibrary of human scFv's.

[0143] EXAMPLE 3—K_(off) determination for scFv fragments binding toCEA.

[0144] EXAMPLE 4—Selection of phage which bind to the mouse anti-CEAantibody from a large library of human scFv's.

[0145] EXAMPLE 5—Marker-ligand-dependent biotinylation of aCEA-expressing cell type.

[0146] EXAMPLE 6—Marker-ligand dependent biotinylation of CEA.

[0147] EXAMPLE 7—Selection of human E-selectin-binding phage from alarge library of human scFv's.

[0148] EXAMPLE8—Selection of novel anti-TGFβ1-binding phage using anexisting anti-TGFβ1-specific scFv.

[0149] EXAMPLE 9—Selection of anti-chemokine receptor phage using achemokine ligand to guide selection.

[0150] EXAMPLE 10—Selection of anti-chemokine receptor phage usinglight-activated streptavidin and the receptor ligand to guide.

[0151] EXAMPLE 11—Selection of phage antibodies to two different cellsurface adhesion molecules using a biotinylated ligand which binds toboth to guide selection.

[0152] EXAMPLE 12—Measurement of the distance over which signal transferusing biotin tyramine may occur.

[0153] EXAMPLE 13—Step-back selection to isolate phage antibodies whichinhibit ligand binding.

[0154] EXAMPLE 14—Biotin tyramine selection in solution using a peptidephage library.

[0155] EXAMPLE 15—Characterisation of clones which bind to CD4+ cells,but not to the chemokine receptor CC-CKR5, by Western blotting and ICC.

[0156] EXAMPLE 16—Demonstration of the use of signal transfer selectionto identify novel protein-protein interactions.

[0157] EXAMPLE 17—Biotinylation of CD4E1 phage on the cell surface usingMIP-1α to direct the biotinylation.

[0158] EXAMPLE 18—Use of biotin tyramine as a signal amplificationreagent in flow cytometry.

[0159] EXAMPLE 19—Iteration of biotin tyramine treatment to give furthersignal enhancement.

[0160] EXAMPLE 20—Use of biotin tyramine to specifically biotinylatesubpopulations of cells to allow their subsequent purification.

[0161] EXAMPLE 21—Biotinylation of phase particles in solution tovalidate biotin-tyramine preparations.

EXAMPLE 1 RECOVERY OF CEA-BINDING PHAGE FROM SURFACE OF CELLS EXPRESSINGCEA IN THE PRESENCE OF A MARKER ANTI-CEA MOUSE MAB.

[0162] a. Purification of CEA-binding phage

[0163] CEA6 is a CEA specific scFv isolated from a large scFv phagedisplay library by panning on human CEA (Vaughan et al 1996). OP1 is acontrol scFv which recognises a 16 residue. peptide and does not bind toCEA. Phagemid particles expressing CEA6 or OP1 scFv's as a fusionproteins with the phage gIII protein were isolated as follows. 500 mlprewarmed (37° C.) 2YTAG (2TY media supplemented with 100 μg/mlampicillin and 2% glucose) in a 2 l conical flask was inoculated withapproximately 3×10¹⁰ cells from a glycerol stock (−70° C.) of CEA6- orOP1 -phagemid. The culture was grown at 37° C. with good aeration untilthe OD 600 nm reached 0.8. M13K07 helper phage (Stratagene) was added tothe culture to a multiplicity of infection (moi) of approximately 10(assuming that an OD 600 nm of 1 is equivalent to 5×10⁸ cells per ml ofculture. The culture was incubated stationary at 37° C. for 15 minutesfollowed by 45 minutes with light aeration (200rpm) at the sametemperature. The culture was centrifuged and the supernatant drainedfrom the cell pellet. The cells were resusupended in 500 ml 2TYAK (2YTmedia supplemented with 100 μg/ml ampicillin and 50 mg/ml kanamycin),and the culture incubated overnight at 30° C. with good aeration(300rpm) Phage particles were purified and concentrated by threepolyethylene glycol (PEG) precipitations (Sambrook, J., Fritsch, E. F.,and Maniatis, T. (1990). Molecular Cloning—A Laboratory Manual. ColdSpring Harbour, N.Y.) and resuspended in PBS to 10¹² transducing units(tu)/ml.

[0164] b. Preparation of HeLa-CEA cell slides.

[0165] CEA-expressing HeLa cells were grown to confluence in DMEMsupplemented with 10% fetal calf serum on 16 chamber slides (Nunc). Thecells were fixed with acetone for 10 minutes, dried and stored at −70 C.

[0166] c. Biotinylation of tyramine.

[0167] An equimolar amount of tyramine (Sigma) was allowed to react withNHS-LC-biotin in 50mM borate buffer, pH 8.8. The reaction was carriedout at room temperature overnight in the dark with rotation. Thebiotinylated tyramine (BT) was filtered through a 0.45 μM filter,aliquotted and stored at −70 C .

[0168] d. Biotinylation of phase binding in close proximity to the Mab.

[0169] HeLa-CEA slides were incubated overnight at 4° C. with 100 μlphage in the presence or absence of an anti-CEA mouse Mab (Zymed) at arange of dilutions from 1:100 to 1:10000 in 3% marvel PBS (MPBS). Phageimput values were around 5×10¹¹ per ml for CEA-purified phage. Controlincubations were carried out in parallel using a phage preparation ofOP1 in the presence of the anti-CEA Mab. 100 μl of phage were used perchamber of the slide. Slide chambers were washed 3 times in PBScontaining 0.1% Tween 20 (PBST), followed by 3 washes with PBS. Eachwash was left for 2 minutes before being changed. 100 μl of a goatanti-mouse HRP second antibody (Pierce) was then added at a dilution inMPBS of 1:2500 and incubated for 1 hour at room temperature. Controlincubations were carried out for the same length of time incubating withPBS alone. Washing was carried out as before and 100 μl of BT in 50mMTris-HCl pH 7.4 with 0.03% H₂O₂ was added to each slide chamber for 10minutes at room temperature. Control incubations were carried out asabove, but with the omission of the BT. Chambers were washed as aboveand phage were then eluted using 200 μl triethylamine (TEA). TEA wasneutralised with 100 μl of 1M Tris-HCl pH 7.4. 10 μl of this elutedphage was used to directly infect an exponentially growing culture of Ecoli TG1. Infected cells were grown for 1 hour at 37° C. with lightaeration in 2YT broth, and then plated on 2TYAG medium. A series ofdilutions of bacteria were plated out and incubated at 30° C. overnight.Colony counts gave the phage titre. The results are shown in Table 1.

[0170] e. Capture of biotinylated phage on streptavidin-coated magneticbeads.

[0171] 20 μl of streptavidin-coated magnetic beads (Dynal) were takenout of solution using a magnet and blocked for 2 hours at roomtemperature on a rotating platform with 1 ml of 3% MPBS. Beads werepelleted using a magnet and 150 μl of eluted phage with 30 μl of 15%MPBS were then added to the blocked beads and rotated for 15 minutes atroom temperature. Beads were pelleted, washed 3 times in PBST and 3times in PBS. The beads were resuspended in a final volume of 100 μlPBS. Half of this was taken and used to directly infect 1 ml of anexponentially growing culture of E coli TG1. Infected cells were grownfor 1 hour at 37° C. with light aeration in 2YT broth, and then platedon 2TYAG medium. A series of dilutions of bacteria were plated out andincubated at 30° C. overnight. Colony counts gave the phage titre. Theresults are shown in Table 1.

[0172] 2. Summary of the results—enhanced recovery of CEA-binding phageusing signal transfer selection followed by streptavidin capture.

[0173] Incubations of CEA6 purified phage on slides coated with CEAtransfected HeLa cells were carried out under a range of differentconditions. Phage imput, primary Mab dilution, presence or absence ofHRP-conjugated second antibody and presence or absence of BT were allexamined. OP1, a non-CEA-specific phagemid which had been selected on a16 residue peptide was also included. The data are shown in Table 1.

[0174] CEA6 phage incubated in the presence of primary Mab,anti-mouse-HRP conjugated second antibody and BT consistently gave thehighest number of phage recovered on the streptavidin-coated magneticbeads. When BT was omitted the number of phage recovered fell by16-fold, and when the primary Mab was omitted phage recovery was reducedby 8-fold. Absence of the HRP conjugated Mab resulted in a 6-foldreduction in phage recovery supporting the conclusion that biotinylationof CEA6 phage is driven by the presence of the Mab-HRP complex. Thisalso demonstrates that only a small proportion of phage are bindingnon-specifically to the Dynal beads in the absence of BT. Somenon-site-specific biotinylation of phage must be occurring since therecovery of phage in the presence of BT, but absence of primary Mab isgreater than the recovery when BT is omitted. Absence of theHRP-antibody conjugate has a simlar effect on the number of phagerecovered compared with absence of the primary Mab. This suggests thatthe secondary Mab is binding specifically to the primary Mab and giveslittle background binding to the cells themselves. The non-CEA-specificphage gave similar levels of biotin-phage recovery as those seen in theabsence of the primary anti-CEA Mab, again suggesting a low level ofnon-site-specific phage biotinylation.

[0175] Overall the results provide an exemplary demonstration of how anexisting Mab raised to a protein of interest can be used to guidecatalysis of biotin deposition onto phage binding the protein ofinterest in the same vicinity as that Mab.

EXAMPLE 2 SELECTION OF CEA-BINDING PHAGE FROM A LARGE LIBRARY OF HUMANSCFV'S

[0176] Antibody repertoire

[0177] The following antibody repertoire was used:

[0178] Large single chain Fv library derived from lymphoid tissuesincluding tonsil, bone marrow and peripheral blood lymphocytes.

[0179] Polyadenylated RNA was prepared from the B-cells of variouslymphoid tissues of 43 non-immunised donors using the “Quickprep mRNAKit” (Pharmacia). First-strand cDNA was synthesized from mRNA using E“First-strand cDNA synthesis” kit (Pharmacia) using random hexamers toprime synthesis. V-genes were amplified using family-specific primersfor VH, V_(K) and Vλ genes as previously described (Marks et al., (1991)J. Mol. Biol. 222:581-597) and subsequently recombined together with the(Gly₄, Ser)₃ scFv linker by PCR assembly. The VH-linker-VL antibodyconstructs were cloned into the Sfi I and Not I sites of the phagemidvector, pCANTAB 6. Ligation, electroporation and plating out of thecells was as described previously (Marks et al, supra). The library wasmade ca. 1000× larger than that described previously by bulking up theamounts of vector and insert used and by performing multipleelectroporations. This generated a scFv repertoire that was calculatedto have ca. 1.3×10¹⁰ individual recombinants which by Bst NIfingerprinting were shown to be extremely diverse.

[0180] a. Induction of phage antibody library

[0181] The phage antibody repertoire above was selected for antibodiesto CEA. The ‘large’ scFv repertoire was treated as follows in order torescue phagemid particles. 500 ml prewarmed (37° C.) 2YTAG (2YT mediasupplemented with 100 μg/ml ampicillin and 2% glucose) in a 2 l conicalflask was inoculated with approximately 3×10¹⁰ cells from a glycerolstock (−70° C.) culture of the library. The culture was grown at 37° C.with good aeration until the OD600 nm reached 0.7 (approximately 2hours). M13K07 helper phage (Stratagene) was added to the culture to amultiplicity of infection (moi) of approximately 10 (assuming that anOD600 nm of 1 is equivalent to 5×10⁸ cells per ml of culture). Theculture was incubated stationary at 37° C. for 15 minutes followed by 45minutes with light aeration (200 rpm) at the same temperature. Theculture was centrifuged and the supernatant drained from the cellpellet. The cells were resuspended in 500 ml 2YTAK (2YT mediasupplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin), and theculture incubated overnight at 30° C. with good aeration (300 rpm).Phage particles were purified and concentrated by three polyethyleneglycol (PEG) precipitations (Sambrook, J., Fritsch, E. F., & Maniatis,T. (1990). Molecular Cloning—A Laboratory Manual. Cold Spring Harbour,N.Y.) and resuspended in PBS to 10¹² transducing units (tu)/ml(ampicillin resistant clones).

[0182] b. Selection of CEA-binding phage from a large non-immunisedphase display library using catalysed enzyme reporter depositionfollowed by streptavidin capture.

[0183] i. First round of selection

[0184] Two rounds of selection using phage prepared from a largenon-immunised human scFv library were carried out on slides ofCEA-expressing HeLa cells. 5×10¹¹ phage were allowed to bind to thecells in the presence or absence of an anti-CEA mouse Mab (Zymed) at adilution of 1:100 in MPBS in a total volume of 100 μl, at 4° C.overnight. Slides were washed Three times in PBST followed by threetimes in PBS. A secondary anti-mouse hydrogen-peroxidase-conjugatedantibody which recognised the primary mouse anti-CEA antibody was thenincubated on the sections at a dilution of 1:2500 in MPBS in a totalvolume of 100 μl at room temperature for 1 hour. Washing was carried outas before and 100 μg of biotinylated-tyramine in 50 mM Tris-HCl pH 7.4with 0.03% H₂O₂ was added to each slide chamber for 10 minutes at roomtemperature. Chambers were washed as above and phage were eluted using200 μl triethylamine (TEA). TEA was neutralised with 100 μl of 1MTris-HCl pH 7.4.

[0185] ii. Assessment of the total number of phage binding to theHeLa-CEA cells

[0186] 10 ml of this eluted phage was used to directly infect anexponentially growing culture of E coli TG1 with light aeration in 2TYbroth at 37° C. for 1 hour. Infected TG1s were plated on 2TYAG medium in243 mm×243 mm dishes (Nunc). Dilutions of infected TG1s were also platedout and incubated at 30° C. overnight. Colony counts gave the phageoutput titre.

[0187] iii. Recovery of biotinylated phase on streptavidin-coatedmagnetic beads

[0188] 20 μl of streptavidin-coated magnetic beads (Dynal) were takenout of solution using a magnet and blocked for 2 hours at roomtemperature on a rotating platform with 1 ml of 3% MPBS. Beads werepelleted and 150 μl of eluted phage with 30 μl of 15% MPBS were thenadded to the blocked beads and rotated for 15 minutes at roomtemperature. Beads were pelleted, washed 3 times in 1 ml PBST and 3times in 1 ml PBS. The beads were resuspended in a final volume of 100μl PBS. 50 μl of this was taken and used to directly infect 1 ml of anexponentially growing culture of E. coli TG1 at 37° C. for 1 hour withlight aeration in 2TYAG medium. Infected TG1s were plated on 2TYAGmedium in 243 mm×243 mm dishes (Nunc). Dilutions of bacteria were alsoplated out and incubated at 30° C. overnight. Colony counts gave thephage output titre.

[0189] iv. Second round of selection

[0190] Colonies were scraped off the 243mm×243mm plates into 3 ml of 2TYbroth and 15% (v/v) glycerol added for storage at −70C. Glycerol stocksolutions from the first round of selection of the repertoire on theHeLa-CEA cells were rescued using helper phage to derive phagemidparticles for the second round of selection. Phagemid particles wererescued from both first round selections carried out in the presence orin the absence of the marker anti-CEA Mab. 250 μl of glycerol stock wasused to inoculate 50 ml 2YTAG broth, and incubated in a 250 mL conicalflask at 37° C. with good aeration until the OD600nM reached 0.7(approximately 2 hours). M13K07 helper phage (moi=10) was added to theculture which was then incubated stationary at 37° C., for 15 minutesfollowed by 45 minutes with light aeration (200 rpm) at the sametemperature. The culture was centrifuged and the supernatant drainedfrom the cell pellet. The cells were resuspended in 50 ml prewarmed2YTAK, and the culture incubated overnight at 30° C. with good aeration.Phage particles were purified and concentrated by PEG precipitation(Sambrook et al., 1990) and resuspended in PBS to 10¹³ tu/ml.

[0191] Phage recovered from the selection in the presence of theanti-CEA mouse Mab underwent a second round selection with either noMab, or with a 1:100, or a 1:1000 dilution of the anti-CEA Mab. Phagerecovered from the first round of selection in the absence of theanti-CEA Mab underwent a second round of selection, again in the absenceof the anti-CEA Mab. The selections were carried out on the HeLa-CEAcells as described for the first round of selection. The total numbersof phage present in the eluates and recovered by streptavidin captureare shown in Table 2.

[0192] The total number of phage recovered on the magnetic beads afterthe first round of selection was comparable either in the presence orabsence of Mab. At round two of the selection the total number ofrecovered phage had dropped to around one tenth of the value from roundone. It was, however, notable that the number of phage recovered aftertwo rounds of selection in the presence of Mab was around 7-fold higherthan that recovered after two rounds of selection without the Mab beingpresent. When one round with Mab present was followed by one roundwithout the Mab the number of recovered phage was around half of thatseen after two rounds of selection with the Mab. Ten-fold dilution ofthe Mab at round 2 of the selections slightly reduced the number ofphage recovered on the Dynal beads (by 12%).

[0193] c. Growth of single selected clones for immunoassay

[0194] Individual colonies from the first and second round selectionswere used to inoculate 100 μl 2YTAG into individual wells of 96 welltissue culture plates (Corning). Plates were incubated at 30° C.overnight with moderate shaking (200 rpm). Glycerol to 15% was added toeach well and these master plates stored at −70° C. until ready foranalysis.

[0195] d. Soluble ELISA to identify anti-CEA scFv

[0196] Cells from the master plates were used to inoculate fresh 96 welltissue culture plates containing 100 μl 2YTAG per well. These plateswere incubated at 30° C. for 8 hours then centrifuged at 2000 rpm for 10min and the supernatant eluted. Each cell pellet was resuspended in 100μl 2YTA containing 10 mM IPTG and incubated at 30° C. overnight.

[0197] Each plate was centrifuged at 2000 rpm and the 100 μl supernatantfrom each well recovered and blocked in 20 μl 18% M6PBS stationary atroom temperature for 1 hour. Meanwhile, flexible microtitre plates whichhad been blocked overnight stationary at 37° C. with either 100 μl 0.5μg/ml CEA in dH₂O or 100 μl dH₂O alone, were washed 3 times in PBS andblocked for 2 h stationary at room temperature in 3MPBS. These plateswere then washed three times with PBS and 50 μl preblocked soluble scFvadded to each well of both the CEA-coated or uncoated plate. The plateswere incubated stationary at 37° C. for 1 h after which the scFvsolutions were poured off. The plates were washed by incubating for 2min in PBST three times followed by incubating for 2 min in PBS threetimes, all at room temperature.

[0198] To each well of both the CEA-coated and the uncoated plate, 100μl of a 1 in 200 dilution of the anti-myc tag murine antibody 9E10(Munro, S. & Pelham, H. R. B. (1986)Cell 46, 291-300) in 3MPBS was addedand the plates incubated at 37° C. stationary for 1 h. Each plate waswashed as described above and 100 μl of a 1 in 5000 dilution goatanti-mouse alkaline phosphatase conjugate (Pierce) in 3MPBS added andincubated stationary at 37° C. for 1 h. Plates were washed as describedabove followed by two rinses in 0.9% NaCl. Alkaline phosphatase activitywas visualised using the chromagenic substrate pNPP (Sigma). Theabsorbance signal generated by each clone was assessed by measuring theoptical density at 405 nm (pNPP) using a microtitre plate reader. Cloneswere chosen for further analysis if the ELISA signal generated on theCEA-coated plate was at least double that on the uncoated plate. Thenumber of clones screened from each round of selection and the number ofCEA positives are shown in Table 3.

[0199] e.Sequencing of anti-CEA ScFv Antibodies

[0200] The nucleotide sequences of the anti-CEA antibodies weredetermined by first using vector-specific primers to amplify theinserted DNA from each clone. Cells from an individual colony on a 2YTAGagar plate were used as the template for a polymerase chain reaction(PCR) amplification of the inserted DNA using the primers pUC19reverseand fdtetseq. Amplification conditions consisted of 30 cycles of 94° C.for 1 min, 55° C. for 1 min and 72° C. for 2min, followed by 10 min at72° C. The PCR products were purified using a PCR Clean-up Kit (Promega)in to a final volume of 50 μl H20. Between 2 and 5 μl of each insertpreparation was used as the template for sequencing using the TaqDye-terminator cycle sequencing system (Applied Biosystems). The primersmycseq10 and PCR-L-Link were used to sequence the light chain of eachclone and PCR-H-Link and pUC19reverse to sequence the heavy chain.

[0201] f. Sequence of the initial CEA-specific scFv antibodies

[0202] Twelve different CEA specific antibodies were isolated from theselections. Each clone name and its heavy and light chain germline isgiven below. The signal transfer method of selection is capable ofgenerating a diverse panel of anti-CEA antibodies. None of theseantibodies were isolated from experiments in which panning of the largescFv library was carried out directly on purified CEA, suggesting thatsignal transfer selection provides a way of accessing different antibodyspecificities from the library. CLONE VH GERMLINE VL GERMLINE SS1A4 VH4DP71 VLambda2 DPL11 SS1A11 VH4 DP71 VLambda2 DPL11 SS1G12 VH4 DP71VKappa1 L12a SS22A4 VH4 DP79 VLambda1 DPL5/2 SS22A8 VR4 DP63 VLambda3DPL16 SS22B7 VH4 DP79 VLambda1 DPL5/2 SS22B1 VH2 V11-5b VLambda1 DPL2SS22D12 VH3 V343 VLambda1 DPL2 SS22E4 VH2 DP28 Vkappa1 DPK8 SS21B1 VH4DP70 Vkappa1 DPK4 SS21B7 VH1 DP71 Vlambda3 DPL16 SSDS1 VH4 DP78 Vlambda3DPL16

EXAMPLE 3 K_(OFF) DETERMINATION FOR SCFV FRAGMENTS BINDING TODESIALYLATED CEA.

[0203] a. K_(off) determination by surface plasmon resonance

[0204] The K_(off)'S for binding to CEA of the scFv fragments describedin Example 2 were determined using desialylated CEA coupled to a CM5sensor chip. 100 μg of CEA was resuspended in 0.1 M sodium acetatebuffer pH 4.0 and desialylated using 1.375mU sialidase (Sigma). This wasincubated for 4 hours at 37° C. with occasional shaking. Thedesialylated CEA was then oxidised using 1 unit of galactose oxidase per500 μg of CEA in 10mM phosphate buffer pH 7.0. This was incubated for 2hours at 36° C. and desalted into 10 mM sodium acetate buffer pH 4.0.The CEA was then immobilised onto the sensor chip using the aldehydegroup. 15 μl EDC/NHS coupling agent (Pierce) was passed over the chip ata flow rate of 5 μl/min. 35 μl of 5mM hydrazine in water was then passedover the chip, followed by 35 μl of ethanolamine. 4 μl of 60 μg/mltreated CEA was passed over the chip at a flow rate of 2 μl/min followedby 40 μl of 0.1 M sodium cyanoborohydride in 0.1 M acetate buffer pH 4.0at a flow rate of 5 μl/min. Approximately 1500RU (resonance units) ofCEA was bound using this method. 5000RU and 800RU CEA chips were madeusing this procedure.

[0205] K_(off)'s were calculated using the Bia-Evalution software(Pharmacia)—Saturation of the chip with purified scFv was demonstratedfor each sample before K_(off) was measured. Results are shown in table4. The range of K_(off)'s of the selected antibodies suggests thatrecovery is dependent on the exact site of binding of the phageantibodies rather than the affinity of the interaction, as is the casewith traditional selection methods. Signal transfer selection is,therefore, a route to obtaining a population of antibodies of diversesequences and affinities which would not normally be obtained by otherselection procedures.

EXAMPLE 4 SELECTION OF PHAGE WHICH BIND TO THE MOUSE ANTI-CEA ANTIBODYFROM A LARGE LIBRARY OF HUMAN SCFV'S

[0206] The antibody repertoire used here and the method of phageinduction was the same as that described in Example 2. The selectionsassayed were the same as those described in Example 2.

[0207] a. Growth of single selected clones for immunoassay

[0208] Individual colonies from the first and second round selectionswere used to inoculate 100 μl 2YTAG into individual wells of 96 welltissue culture plates (Corning). Plates were incubated at 30° C.overnight with moderate shaking (200 rpm). Glycerol to 15% was added toeach well and these master plates stored at −70° C. until ready foranalysis.

[0209] b. Soluble ELISA to identify anti-scFv

[0210] Cells from the master plates were used to inoculate fresh 96 welltissue culture plates containing 100 μl 2YTAG per well. These plateswere incubated at 30° C. for 8 hours then centrifuged at 2000 rpm for 10min and the supernatant eluted. Each cell pellet was resuspended in 100μl 2YTA containing 10 mM IPTG and incubated at 30° C. overnight.

[0211] Each plate was centrifuged at 2000 rpm and the 100 μl supernatantfrom each well recovered and blocked in 20 μl 18% M6PBS stationary atroom temperature for 1 hour. Meanwhile, flexible microtitre plates whichhad been incubated overnight stationary at 37° C. with either 50 μl 0.1μg/ml of anti-CEA mouse Mab in PBS or 50 μl PBS alone, were washed 3times in PBS and blocked for 2 h stationary at room temperature in3MPBS. These plates were then washed three times with PBS and 50 μlpreblocked soluble scFv added to each well of both the anti-CEA-mouseMab-coated or uncoated plate. The plates were incubated stationary at37° C. for 1 h after which the scFv solutions were poured off. Theplates were washed by incubating for 2 min in PBST three times followedby incubating for 2min in PBS three times, all at room temperature.

[0212] To each well of both the mouse Mab-coated and the uncoated plate,100 μl of a 1 in 200 dilution of biotinylated anti-myc tag murineantibody 9E10 (Munro, S. & Pelham, H. R. B. (1986) Cell 46, 291-300) in3MPBS was added and the plates incubated at 37° C. stationary for 1 h.Each plate was washed as described above. Plates were then incubatedwith alkaline-phosphatase-streptavidin complex (DAKO) diluted 1:1000 indH₂O. Plates were washed as described above followed by two rinses in0.9% NaCl. Alkaline phosphatase activity was visualised using thechromogenic substrate pNPP (Sigma). The absorbance signal generated byeach clone was assessed by measuring the optical density at 405 nm(pNPP) using a microtitre plate reader. Clones were-scored for positivebinders for the anti-CEA mouse Mab if the ELISA signal generated on theCEA-coated plate was at least double that on the uncoated plate.

[0213] Clones from the various round 2 selections were screened foranti-CEA mouse Mab binding (Table 5). 12.5% of clones which had comethrough two rounds of 1:100 Mab selections were found to bind the Mab.No Mab binders were present in the population which came through tworounds of selection with no Mab present. This demonstration that some ofthe recovered phage recognise the anti-CEA mouse Mab is evidence forgeneral biotinylation of any phage binding in close proximity of theanti-mouse HRP secondary antibody and hence is evidence of thesite-specific nature of the interaction.

EXAMPLE 5 MARKER-LIGAND-DEPENDENT BIOTINLATION OF A CEA-EXPRESSING CELLTYPE.

[0214] a. Biotinylation by biotin tyramine of the HeLa-CEA expressingcells grown on slides.

[0215] Thawed HeLa-CEA slides which had been rehydrated in PBS for 10minutes at room temperature were incubated for 15 minutes withstreptavidin in PBS at 10 μg/ml. Slides were washed four times in PBSand then incubated for 15 minutes with in PBS at 10 μg/ml. Slides werewashed 4 times in PBS and then incubated in block consisting of 1% BSAPBS containing 10% normal mouse serum for 30 minutes. Block was removedand the slides then incubated with CEA6 purified phage (as described inExample 1) at approximately 1×10¹⁰ per ml in 1%PBS-BSA overnight at 4°C. Control slides were incubated under the same conditions with purifiedfluorescein-binding phage which do not recognise CEA. Slides were movedback to room temperature and washed in PEST for 10 minutes, followed byincubation with 9E10-biotin at 3μg/ml diluted in 1% BSA-PBS for 1 hour.Washing was carried out for 10 minutes in PBST and the slides thenincubated with ABC-HRP (DAKO) diluted 1:100 in PBS for 30 minutes.Slides were either developed at this point or washed three times inPBST, then incubated in either with biotinylated tyramine in 50mMTris-HCl pH 7.4 containing 0.03% H₂O₂ for 10 minutes. Three PBST washeswere carried out and the slides then incubated with the ABC-HRP complexagain for 30 minutes. Slides were developed using carbazole. Carbozolewas prepared freshly by dissolving 9-amino-ethyl-carbozole (Sigma) at 60mg per 25 ml DMF then adding 100 μl of this to 1 ml sodium acetate pH5.2.5 μl of 30% H₂O₂ was then added and 100 μl of this mix added to eachslide chamber. Development was left for 20 minutes and then thecarbazole washed off with dH₂O.

[0216] Slides incubated with the CEA6 phage but without thebiotin-tyramine amplification step showed faint red staining in regionsblebbing from the HeLa-CEA cell surfaces, whereas the slides treatedwith the anti-fluorescein phage showed no such staining. Slidesincubated with CEA6 phage and then subjected to a round of biotintyramine treatment shown significantly stronger staining of the regionsof CEA, demonstrating that proteins present in the region of CEA6 phagebinding had been biotinylated and were able to amplify the colourreaction due to recruitment of more ABC-HRP complex.

EXAMPLE 6 MARKER-LIGAND DEPENDENT BIOTINYLATION OF CEA.

[0217] i. Biotinylation of CEA.

[0218] HeLa-CEA expressing cells grown in chamber flasks were blocked inMPBS for 2 hours at room temperature. 100 μl of an anti-CEA mouse Mabwas then incubated on the slides at a dilution of 1:100 in MPBS for 1hour at room temperature. Control incubations were carried out in MPBSwithout the presence of the anti-CEA Mab. Slides were washed three timesin PEST followed by three washes in PBS. 100 μl of of a goat anti-mouseHRP-conjugated second antibody (Pierce) was then added at a dilution of1:2500 in MPBS and incubated for 1 hour at room temperature. Washing wascarried out as before and 100 μl of biotinylated tyramine in 50mMtris-HCl pH 7.4 with 0.03% H₂O₂ was added to each slide chamber for 10minutes at room temperature. Cells were then scraped off the slides.Cells were pelleted at 600 rpm for 5 minutes and then resuspended in10mM triethanolamine, 1% triton in saline. Cells were left on ice for 10minutes, then cell nuclei were pelleted at 13000 rpm in a minifuge for 5minutes at 4° C. Supernatants were added to reducing protein loadingbuffer and run on 10-15% SDS gradient PHAST gels. Protein weretransferred to Hybond C extra (Amersham) membranes using the PHASTsystem programme at 70° C. for 30 minutes. Membranes were blocked for 2hours in MPBS and incubated for 1 hour at room temperature with either astrepavidin-HRP complex, or an anti-CEA mouse Mab. Blots probed with theanti-CEA mab were washed three times in PEST followed by three washes inPBS, then incubated with an anti-mouse-HRP-conjugated antibody at adiltuion of 1:2500 in MPBS for 1 hour at room temperature. Blots werewashed as before and developed using the ECL (Amersham) detection kit.

[0219] The western blot probed with streptavidin-HRP conjugate showedthe presence of one major high molecular weight band in the Hela-CEAcells treated with anti-CEA, anti-mouse-HRP and then biotinylatedtyramine. This band was shown to be reactive with an anti-CEA Mab. Theband was a higher molecular weight than that theoretically anticipatedfor CEA, probably due to the many carbohydrate groups on CEA whichresult in retarded migration of the CEA. Two other biotinylated minorbands could be detected at round the expected size for a Mab orconjugated Mab. These bands could potentailly be biotinylated forms ofthe anti-CEA Mab and the anti-mouse-HRP conjugate. No other clear bandscould be seen on the blot, although some some less specificbiotinylation may be indicated by the presence of a high molcular weightsmear after a long exposure (20 minutes) of the blot to ECL (Amersham)film in the lane corresponding to the Hela-CEA cells which were treatedwith both antibodies and the BT. Control lanes, in which treatment ofthe cells with BT or with the anti-CEA Mab was omitted, showed noevidence of biotinylation. This demonstrates the ability of the biotintyramine system to selectively “tag” proteins binding in close proximityto a marker ligand to allow their detection and facilitate theirpurification.

EXAMPLE 7 SELECTION OF HUMAN ANTI-E-SELECTIN-BINDING PHAGE FROM A LARGESCFV LIBRARY.

[0220] a. Conjugation of polyclonal anti-E selectin IgG to HRP

[0221] Polyclonal anti-human-E-selectin IgG was obtained from R and DSystems. The conjugation was carried out using a hydrogen peroxidaseconjugation kit supplied by Pierce. 1 mg of maleimide-activated HRP wasconjugated to 100 μg of Mab using the SATA protocol (Pierce). 20 μl of a4 mg/ml SATA solution made up in DMF was added to 100 μl of polyclonalIgG in PBS. This was incubated for 30 min at room temperature, then 100μl of deacetylation solution (Pierce) was added and incubation wascontinued for a further 2 hours at room temperature. Deacetylated IgGwas separated from unreacted and deacetylated SATA on a 5 ml sepharose25 column which had been pre-equilibriated with maleimide conjugationbuffer. 0.5 ml fractions were collected and the majority of the proteinwas collected in fractions 2 and 3. 1 ml of the is deacetylated IgG wasthen added to 1 mg of maleimide-activated HRP and incubated at roomtemperture for 1 hour.

[0222] b. Cell culture

[0223] Human vascular endothelial cells (HUVECs) (Clonetics) werecultured to passage 3 on 24 well plates (Nunc) coated with 1% gelatin.The cells were grown to approximately 80% confluence using EGM medium(Clonetics). HUVEC cells express a low basal level of the adhesionprotein E-selectin.

[0224] c. Selection procedure

[0225] Cells were washed with PBS and the cells were then incubatedovernight at 4° C. in 200 μl of PBS/1% BSA in the presence of 1×10¹²phage prepared from a large non-immunised human scFv library. To oneculture well a 1:20 dilution of the HRP-conjugated polyclonal anti-Eselectin IgG was also added to the phage. Cells were washed three timesin 0.5 ml PBST and three times in PBS. 200 μl of the biotin tyramine mix(as in Example 2 part bi) was added to each well and left for 10 minutesat room temperature. Cells were washed as before and the phage theneluted in 200ml triethylamine (TEA) for 10 minutes at room temperature.The TEA was then neutralised with 100 μl of 1M TrisHCl pH 7.4.

[0226] d. Recovery of biotinylated phage

[0227] 20 μl of streptavidin-coated magnetic beads (Dynal) were takenout of solution using a magnet and blocked for 2 hours at roomtemperature on a rotating platform with 1 ml of 3% MPBS. Beads werepelleted and 300 μl of eluted phage with 60 μl of 15% MPBS were added tothe blocked beads and rotated for 15 minutes at room temperature. Beadswere pelleted, washed three times in 1 ml PBST and three times in 1 mlPBS. The beads were resuspended in a final volume of 100 μl PBS. 50 μlof this was taken and used to directly infect 5 ml of an exponentiallygrowing culture of E coli TG1 at 37° C. for 1 hour with light aerationin 2TYAG medium. Infected TG1 s were plated on 2TYAG medium in 243mm×243mm dished (Nunc). Dilutions of bacteria were also plated out andincubated at 30° C. overnight. Colony counts gave the phage outputtitre.

[0228] Output titres for selections: Total Captured % phage eluate phagecaptured Minus anti-E-sel-HRP   8 × 10⁴  81 0.10 conjugate Plusanti-E-sel-HRP 1.6 × 10⁴ 498 3.11 conjugate

[0229] The percentage of biotinylated phage captured on the beads in thepresence of the HRP-conjugated polyclonal anti-E selectin IgG is around30-fold higher than the percentage captured in the absence of theantibody. This suggests the HRP-anti-E-selectin polyclonal IgG istargeting the biotinylation of E-selectin-specific phage.

[0230] e. Growth of single selected clones for soluble ELISA to identifyanti-E-selectin scFv

[0231] Single colonies were grown up exactly as described in Example 2part c and the ELISAs were carried out as in part d, except that theplates were coated with 1 μg/ml recombinant E selectin (R and DSystems).

[0232] The number of positives screened from each round of selection andthe number of E-selectin positive clones are shown below. No. clonesE-sel + ve % E-sel + ve Minus anti-E sel HRP  95 0 0   conjugate Plusanti-E sel HRP 282 8 2.8 conjugate

[0233] The ELISA results demonstrate the increase in the number ofE-selectin binders selected for in the presence of the polyclonal anti-Eselectin HRP conjugate compared to the selection when this antibody isomitted. This demonstrates that the antibody-HRP conjugate isresponsible for the specific biotinylation of phage binding in closeproximity to it.

EXAMPLE 8 SELECTION OF NOVEL TGFβ1-BINDING PHAGE USING AN EXISTINGANTI-TGFβ1-SPECIFIC scFv.

[0234] 31G9 is a high affinity (1.2×10⁻⁹ M) anti-TGFβ1 -specific scFvwhich was previously isolated from a large human non-immunised scFvphage display library by direct selection of the library on immobilisedTGFβ1. The antibody does not recognise a neutralising epitope of TGFβ1.Investigations were carried out to Assess whether a HRP-conjugate of31G9 could be used in a signal transfer selection to isolate newlineages of phage antibodies which recognise different, potentiallyneutralising epitopes of TGFβ1.

[0235] a. Conjugation of 31G9 scFv to HRP.

[0236] 31G9 was conjugated to maleimide-activated HRP as described inExample 7, part a, except that 300 μg of purified scFv was used in theconjugation reaction.

[0237] b. Preparation of a low density TGF62 1 BiaCore chip.

[0238] 50 μl of NHS/EDC reagent (Pharmacia) was incubated for 30 min atroom temperature on the surface of a CM5 chip. The chip was washed 5times in HBS and 75ng of TGFβ1 in 75 μl of 10mM sodium citrate buffer pH3.6 was then incubated on the chip for 1 hour at room temperature. Thechip was washed 5 times in HBS and then treated with 1M ethanolamine pH8 for 10 min. The chip was stored at 4° C. in HBS. Approximately 40resonance units (RUs) of TGFβ1 were linked to the chip.

[0239] c. Selection procedure.

[0240] i) First round of selection.

[0241] 100 μl of HRP-conjugated 31G9 (approximately 30 μg) was incubatedon the TGFβ1-coupled BiaCore chip for 1 hour at room temperature. Thechip was washed 3 times in PBST and 3 times in PBS and 1×10¹² phageprepared from the human non-immunised library were then incubated on thechip surface for 1 hour at room temperature. The chip was washed asbefore and 100 μl of biotin tyramine mix (as described in Example 2 partbi) was incubated on the chip for 10 min at room temperature. The chipwas washed as before and phage eluted from the chip using 200 μl oftriethylaime TEA. The TEA was neutralised with 100 μl of 1M Tris-HCl pH7.4.

[0242] ii) Recovery of biotinylated phage

[0243] 20 μl of streptavidin-coated magnetic beads (Dynal) were takenout of solution using a magnet and blocked for 2 hours at roomtemperature on a rotating platform with 1 ml of 3% MPBS. Beads werepelleted and 300 μl of eluted phage with 60 μl of 15% MPBS were added tothe blocked beads and rotated for 15 minutes at room temperature. Beadswere pelleted, washed three times in 1 ml PBST and three times in 1 mlPBS. The beads were resuspended in a final volume of 100 μl PBS. 50 μlof this was taken and used to directly infect 5 ml of an exponentiallygrowing culture of E coli TG1 at 37° C. for 1 hour with light aerationin 2TYAG medium. Infected TG1s were plated on 2TYAG medium in 243 mm×243mm dished (Nunc). Dilutions of bacteria were also plated out andincubated at is 30° C. overnight. Colony counts gave the phage outputtitre.

[0244] iii) Second round of selection.

[0245] Colonies were scraped off the 243 mm×243 mm plates into 3 ml of2TY broth and 15% (v/v) glycerol added for storage at −70C. Glycerolstock solutions from the first round of selection of the repertoire onthe TGFβ1-BiaCore chip were rescued using helper phage to derivephagemid particles for the second round of selection. 250 μl of glycerolstock was used to inoculate 50 ml 2YTAG broth, and incubated in a 250 mLconical flask at 37° C. with good aeration until the OD600nM reached 0.7(approximately 2 hours). M13K07 helper phage (moi=10) was added to theculture which was then incubated stationary at 37° C. for 15 minutesfollowed by 45 minutes with light aeration (200 rpm) at the sametemperature. The culture was centrifuged and the supernatant drainedfrom the cell pellet. The cells were resuspended in 50 ml prewarmed2YTAK, and the culture incubated overnight at 30° C. with good aeration.Phage particles were purified and concentrated by PEG precipitation(Sambrook et al., 1990) and resuspended in PBS to 10¹³ tu/ml.

[0246] The second round of selection and capture of biotinylated phageon the. TGFβ1-BiaCore chip was carried out exactly as the first round.The phage output titres are shown below. Strepavidin captured Totaloutput output % captured Round 1 2.5 × 10⁷   5 × 10⁵ 2   Round 2   6 ×10¹⁰ 1.8 × 10⁵ 0.5

[0247] d. Growth of single selected clones for soluble ELISA to identifyanti-TGFβ1 scFv

[0248] Single colonies were grown up exactly as described in Example 2part c and the ELISAs were carried out as in part d, except that theplates were coated with 0.2 μg/ml recombinant TGFβ1 (R and D Systems).The 192 clones from the second round of selection were screened by ELISAand 26 were found to be TGFβ1 positive (13.5%).

[0249] e sequencing of anti-TGFβ1scFv Antibodies

[0250] The nucleotide sequences of the anti-TGFβ1 antibodies weredetermined by first using vector-specific primers to amplify theinserted DNA from each clone. Cells from an individual colony on a 2YTAGagar plate were used as the template for a polymerase chain reaction(PCR) amplification of the inserted DNA using the primers pUC19reverseand fdtetseq. Amplification conditions consisted of 30 cycles of 94° C.for 1 min, 55° C. for 1 min and 72° C. for 2min, followed by 10 min at72° C. The PCR products were purified using a PCR Clean-up Kit (Promega)in to a final volume of 50 μl H₂O. Between 2 and 5 μl of each insertpreparation was used as the template for sequencing using the TaqDye-terminator cycle sequencing system (Applied Biosystems). The primersmycseq10 and PCR-L-Link were used to sequence the light chain of eachclone and PCR-H-Link and pUC19reverse to sequence the heavy chain

[0251] Sequencing revealed that a total of six different anti-TGFβ1antibodies had been isolated by the signal transfer selection methodusing the 31G9-HRP conjugate to target the site specific biotinylation.These six antibodies were of VH germlines different from that of 31G9,as shown below. CLONE VH GERMLINE VL CERMLINE ST3 VH3 DP53 VLambda2DPL11 ST6 VH3 DP53 VLambda3 DPL16 ST10 VH3 DP53 VLambda3 DPL16 ST14 VH3DP53 VLambda2 DPL12 ST19 VH3 DP53 VKappa1 DPK9 ST21 VH3 DP53 VLambda2DPL12 31G9 VH3 DP49 VKappa1 DPK9

[0252] All the clones selected had the same VH3 DP53 germline pairedwith a variety of VL gene segments. ST6 and ST10 had the same VLgermline and differed from each other at a single amino acid residue inVL CDR2, ST14 and ST21 also had the same VL germline but differed fromeach other at a single amino acid residue in VL CDR3. None of theselected clones had the same VH as 31G9. Clone ST19 had the samegermline VL as 31G9 with a single amino acid change in VL FR2.

[0253] Overall this demonstrates the ability of the signal tansferselection technique to select away from an undesired antigenic epitopeand generate new lineages of phage antibodies which may have alteredspecificities.

EXAMPLE 9 SELECTION OF ANTI-CHEMOKINE RECEPTOR PHAGE USING A CHEMOKINELIGAND TO GUIDE SELECTION.

[0254] The chemokine receptor CC-CKR5 is a co-receptor for macrophagetropic HIV-1 strains which i6 expressed on CD4⁺ lymphocytes. CC-CKR-5responds to a number of chemokines, including macrophage inflammatoryprotein (MIP)-1α. MIP-1α also binds to other chemokine receptors,including CC-CKR1 and CC-CKR4. MIP-1α may be used to guide signaltransfer selection of phage antibodies or other phage displayed proteinswhich bind to the CC-CKR5 receptor.

[0255] a. Preparation of human CD4+ cells from blood.

[0256] Mononuclear cells were prepared from a 50 ml buffy coat usingFicoll-Paque (Pharmacia) density gradient centrifugation (600 g for 20min at 20° C.). CD4⁺ cells were then isolated from the 1.5×10⁸ recoveredcells using a Biotex CD4 column, following the manufacturer'sinstructions, although PBS/2% foetal calf serum (FCS) was usedthroughout. Eluted cells were pelleted at 600 g for 5 min andresuspended in 300 μl PBS/2% FCS. 8.3×10⁶ cells were recovered usingthis procedure. The recovered cells were analysed by flow cytometry andapproximately 59% of the cells were found to be CD4⁺.

[0257] b. Selection procedure and capture of biotinylated phage.

[0258] 1×10⁵ CD4⁺ lymphocytes were incubated with 2×10¹² phage preparedfrom the 1.4×10¹⁰ scFv phage display library in either the presence orabsence of biotinylated MIP-1α (R and D Systems) at a finalconcentration of 375nM. The final volume for each selection was made up40 μl with PBS containing 2% marvel (MPBS). Selections were incubatedfor 14 hr at 4° C. Cells were pelleted by centrifugation at 600 g for 3min, and washed in 1 ml MPBS. A total of three washes were carried out.100 μl of streptavidin-HRP was added at a dilution of 1:1000 in MPBS.This was incubated for 2 hr, then washed as before. Biotin tyramine wasthen added (as Example 2, part bi) in 100 μl of 150mM NaCl/50mM TrisHClpH 7.4 containing 3% H₂O₂ and incubated for 10 min at room temperature.Cells were washed and resuspended in 100 μl TE containing 0.5% triton.Biotinylated phage were captured on 10 μl of MPBS-blockedstreptavidin-coated magnetic beads (Dynal). The beads were washed threetimes in 1 ml PBS/0.1% Tween 20 (PBST), then resuspended in 100 μl ofPBS. Phage eluate before and after streptavidin capture were titred byinfection of an exponentially growing culture of E coli TG1 at 37° C.for 1 hr. The numbers of phage recovered from the various selectionprocedures are shown below. Selection Bio-MIP-1α No. phage % phage No.Bio-tyzamine Strep-HRP Captured Captured Eluted Total No. 1   + + 3.7 ×10⁵ 5.9 × 10³ 1.6 2   + − 4.0 × 10⁵ 8.0 × 10² 0.2 3   − + 4.9 × 10⁵ 1.4× 10³ 0.3

[0259] The greatest recovery of biotinylated phage was observed fromCD4⁺ lymphocytes incubated with both the biotinylated MIP-1α and biotintyramine. Omission of either the biotinylated ligand or the biotintyramine resulted in an approximately 5 to 6-fold drop in the percentageof phage recovered from the eluate. These results suggest thebiotinylated MIP-1α is capable of binding the CD4⁺ cells in the presenceof the phage library and directing biotinylation of phage binding aroundit in the presence of HRP and hydrogen peroxide.

[0260] c. Phage ELISA to identify CD4⁺ cell binders, CC-CKR5 transfectedcell binders and CC-CKR5 amino terminal peptide binders.

[0261] Selected phage were analysed by phage ELISA for their ability torecognise CD4⁺ lymphocytes, a CC-CKR5 transfected cell line (provided byM. Parmentier and G. Vassart, University of Brussels) and aBSA-conjugated peptide corresponding to the amino terminal twenty aminoacids of the CC-CKR5 receptor (MDYQVSSPIYDINYYTSEPC). Phage ELISAs werecarried out as follows: individual clones were picked into a 96 welltissue culture plate containing 100 μl 2YTAG. Plates were incubated at37° C. for 6 hours. M13KO7 helper phage was added to each well to an moiof 10 and incubated with gentle shaking for 45 min at 37° C. The plateswere centrifuged at 2000 rpm for 10 min and the supernatant removed.Cell pellets were resuspended in 100 μl 2TYA with kanamycin (50 μg/ml)and incubated at 30° C. overnight. The ELISA was then carried out as forsoluble ELISA (Example 2) except that in place of the 9E10 a goatanti-M13 antibody was used at a dilution of 1:2500, followed by ananti-goat alkaline phosphatase conjugate, also at a dilution of 1:2500.1×10⁵ cells per ELISA well were used and the peptide BSA conjugate wascoated at a concentration of 1 μg/ml.

[0262] 30/95 of the phage selected in the presence of biotin tyramineand MIP-1α recognised CD4⁺ lymphocytes. 11/95 of the phage selected inthe absence of MIP-1α recognised CD4 ⁺ lymphocytes. 13 of the 30 cloneswhich were positive on CD4⁺ cells were also found to be positive on theCC-CKR5 cell line. Of these two clones (RK-1 and RK-2) selected id thepresence of MIP-1α and biotin tyramine were found to be specific for theCC-CKR5 peptide. The clones which do not recognise the CC-CKR5 peptidemay of course recognise other epitopes of CC-CKR5, other MIP-1αreceptors or proteins which are found on the cell surface in closeproximity to MIP-1α is receptors.

[0263] d. Sequencing of RK1 and RP2.

[0264] Sequencing of the two peptide binding clones was carried out asdescribed in Example 2 part f. Clones RK1 and RK2 had identical VL genesegments. VH VH VL VL family segment family segment RK1 VH4 DP67 V13DPL16 RK2 VH4 DP14 V13 DPL16

[0265] e. Western blotting using RK2

[0266] A representative of these peptide-binding clones (RK-2) wastested by western blotting on extracts from a CC-CKR5 transfected cellline and was found to bind to an approximately 35kD band which maycorrespond to CC-CKR5.

[0267] This work describes the use of signal transfer selection toisolate phage antibodies of a desired specificity directly from a largephage library using a ligand of a known binding specificity (MIP-1α) asa marker to guide selection of phage binding in an area around theligand binding site. A proportion of the resultant selected populationhas been shown to be specific for the ligand's receptor (CC-CKR5). Theantibodies generated in this example bind to a seven transmembraneprotein which acts as a co-factor in HIV infection, hence the antibodiesmay have a therapeutic role.

EXAMPLE 10 SELECTION OF ANTI-CHEMOKINE RECEPTOR PHAGE USING

[0268] LIGHT-ACTIVATED STREPTAVIDIN AND THE RECEPTOR LIGAND TO GUIDE.

[0269] As described in Example 9 MIP-1α can be used to guide selectionof antibodies to at least one of its receptors (CC-CRK5). This exampleutilises the same system to demonstrate to ability of light activatiblestreptavidin to be used instead of biotin tyramine in an analogoussignal transfer procedure.

[0270] a. Generation of light activatible streptavidin.

[0271] SAND (sulphosuccinimidyl2-[m-azido-o-nitrobenzamidol]-ethyl-1,3-dithiopropionate, Pierce) is aphotocrosslinking agent which is activated in the visible range(300-460nm ). SAND was linked to streptavidin by mixing 2 mg/mlstreptavidin (Pierce) 7.5mM SAND in PBS. This was incubated in the darkroom at room temperature for 2 hr, then separated on a NAPS column.

[0272] b. Selection procedure

[0273] 1×10⁵ CD4⁺ lymphocytes were prepared as described in Example 9part a and incubated with 2×10¹² phage prepared from the 1.4×10¹⁰ scFvphage dsiplay library in either the presence or absence of biotinylatedMIP-1α (R and D Systems) at a final concentration of 375nM. The finalvolume for each selection was made up 40μl with PBS containing 2 marvel(MPBS). Selections were incubated for 14 hr at 4° C. Cells were pelletedby centrifugation at 600 g for 3 min, and washed in 1 ml MPBS. A totalof three washes were carried out. Cells were then incubated in the darkfor 30 min with 500mM streptavidin-conjugated SAND. Cells were washed asbefore, the exposed to 5 flashes of light from a standard flashgun.Cells were pelleted and resuspended in 100 μl TE containing 0.5% triton.

[0274] c. Captured of streptavidin-linked phage

[0275] The eluate was added to preblocked immunosorb tubed coated with 1ml of 100 μg/ml biotinylated-BSA. After 1 hour the tube was washed 10times in 1 ml PBS. Phage which had been cross-linked to the streptavidinwere eluted in 1 ml PBS containing 28mM b-mercaptoethanol. Phage fromthe total eluate and from the captured population were titred. Thenumbers of phage recovered from the various selection procedures areshown below. Selection phage No. Bio-MIP-1α Strep Total No. Captured %phage SAND Eluted Captured No. 1 + + 8.2 × 10⁴ 54 0.06 2 + − 3.6 × 10³ 00 3 − + 4.4 × 10⁵ 0 0

[0276] Phage were only recovered from the final eluate whenstreptavidin-SAND was included in the selection scheme. In the absenceof this no background phage were recovered. These results deomstrate theability of biotinylated MIP-1α and a light activatible streptavidinmolecule to specifically cross-link streptavidin to phage binding aroundthe site of MIP-1α binding.

[0277] c. Phage ELISA to identify CD4⁺ cell binders, CC-CKR5 transfectedcell binders and CC-CKR5 amino terminal peptide binders.

[0278] Selected phage were analysed by phage ELISA for their ability torecognise CD4⁺ lymphocytes, a CC-CKR5 transfected cell line (provided byM. Parmentier and G. Vassart, University of Brussels) and aBSA-conjugated peptide corresponding to the amino terminal twenty aminoacids of the CC-CKR5 receptor (MDYQVSSPIYDINYYTSEPC). Phage ELISAs werecarried out as described in Example 9.

[0279] 24/54 of the phage selected in the presence of biotin tyramineand MIP-1α recognised CD4⁺ lymphocytes. 15 of the 24 clones which werepositive on CD4⁺ cells were also found to be positive on the CC-CKR5cell line. Of these two clones (RK-3 and RK-4) were found to be specificfor the CC-CKR5 peptide.

[0280] d. Sequencing of RK3 and RK4.

[0281] Sequencing of the two peptide binding clones was carried out asdescribed in Example 2 part f. VH VH VL VL family segment family segmentRK3 VH4 DP14 Vλ3 DPL16 RK4 VH4 DP14 Vλ3 DPL16

[0282] RK1, which was a clone generate by biotin tyramine signaltransfer selection using MIP-1α as a guide molecule was identical toRK-3, with the exception of a single amino acid difference in the VLCDR3. This demonstrates the selectivity of the selection procedures; avirtually identical clone recognising the same CC-CKR5 region can beselected by either biotin tyramine or light activatible-streptavidinsignal transfer selection from a background of 1×10¹⁰ other clones.

EXAMPLE 11 SELECTION OF PHAGE ANTIBODIES TO TWO DIFFERENT CELL SURFACEADHESION MOLECULES USING A BIOTINYLATED LIGAND WHICH BINDS TO BOTH TOGUIDE SELECTION.

[0283] E and P selectin are cell adhesion molecules which are expressedon the surface of human vascular endothelial cells (HUVECs). E and Pselectin are upregulated after stimulation with thrombogenic orinflammatory agents such as TNFα. The ligand for both these selectin hasbeen found to be sialyl Lewis X, and this ligand has been used togenerate antibodies to both of its receptor adhesion molecules in thesame selection.

[0284] a. Stimulation of HUVEC's using TNFα.

[0285] HUVEC'S (grown to passage 5) were stimulated with TNFα at500pg/ml for 4 hours and flow cytometry analysis was carried out toensure that E selectin was up-regulated. After stimulation 43.8 percentof the cells treated gave a fluorescence value greater than 1, whereaswithout stimulation only 2.6 percent of the cells gave fluorescencegreater than 1.

[0286] b. Biotinylation of sialyl Lewis X.

[0287] Sialyl Lewis X (Oxford Glycosystems) was biotinylated usingbiotinylated diaminopyridine (BAP). 1 mg BAP was dissolved in 50 μlpyridine/acetic acid (2:1 v/v) This was added directly to the drycarbohydrate (100 μg) and incubated for 1 hour at 80° C. Theoligosaccharide-BAP adducts were reduced by the addition of 50 μl of 2.1M/l borane dimethylamine in pyridine/acetic acid and vortexed.Incubation was then carried on for a further hour at 80° C.

[0288] c. Selection and capture of biotinylated phase.

[0289] Stimulated HUVEC's were incubated with phage rescued from thelarge non-immunised scFv library. Two rounds of signal transferselections were carried out in the presence or absence of 40 μg ofbiotinylated sialyl Lewis X. Phage were captured on streptavidin-coatedmagnetci beads as described in Example 1 part (e). The number of phagepresent before and after capture was titred. The greatest recovery ofbiotinylated phage was observed from stimulated cells when biotinylatedsialyl Lewis X and biotin tyramine steps were present (1.8% recovery).Omission of either the biotinylated sialyl Lewis X or biotin tyramineresulted in an approximately 10-fold drop in the % of phage recoveredfrom the eluate (both gave 0.2% recovery). These results suggest thatbiotinylated sialyl-Lewis X (with-streptavidin-HRP) is capable ofbinding to the stimulated HUVEC's in the presence of the phage libraryand directing biotinylation of phage binding around the ligand bindingsites.

[0290] d. Soluble ELISA to identify E- and P-selectin binders.

[0291] Recovered phage were examined by soluble ELISAs [as described inExample 2 part (d)] for their binding to E and P selectin. 3.6% of theclones recovered from the first round of selection in the presence ofbiotinylated sialyl Lewis X and biotin tyramine were E selectinpositive. None of the clones tested from selections carried out onunstimulated cells, or in the absence of ligand or biotin tyramine wereE selectin positive. 2.8% of the clones recovered from theHRP-conjugated anti-E selectin IgG selections were found to bind Eselectin, whereas in the absence of the HRP-conjugate no clones werefound to be E selectin positive. From the second round of selection inthe presence of sialyl Lewis X and biotin tyramine the number of clonesfound to be E selectin positive increased to 13.7%.

[0292] P selectin ELISAs were also carried out on the population ofclones selected in the presence of biotinylated sialyl Lewis X andbiotin tyramine on stimulated cells 50 of the E selectin binders werealso found to recognised P selectin, which shares sialyl Lewis X as itsligand. In addition a further 2% were found to be P selectin specific.

[0293] A further 21% of the second round selected population were foundto bind to stimulated HUVEC's by soluble ELISA. The clones found to bindE selectin were sequenced and a diverse population of E selectin binderswere identified. A range of different germline VH's were selected. TheVL's were less diverse; a total of 4 different germline segments wereselected which had common CDR3's.

[0294] These selections demonstrate the ability of a natural ligand fora particular cell surface protein to direct selection of cell surfaceprotein binding clones. A ligand which recognizes more than one cellsurface protein (in this case E and P selectin) can be used to guideselection of antibodies to either of its target proteins.

EXAMPLE 12 MEASUREMENT OF THE DISTANCE OVER WHICH SIGNAL TRANSFER USINGBIOTIN MAY OCCUR.

[0295] The following experiment was designed to assess the distance overwhich biotinylation may occur using HRP and biotin tyramine.Bacteriophage are approximately 1 μm long filaments with three copies ofthe gene 3 protein at one end of the filament. The gene 3 proteinprovides a marker which can be used to localise HRP specifically to oneend of the phage via a mouse anti-gene 3 antibody, followed by ananti-mouse-HRP conjugate. Biotin tyramine and hydrogen peroxide can thenbe added to the tagged phage to allow biotinylation of the phage aroundthe site of the HRP activity. Phage can then be transferred to electronmicroscope grids and labelled with streptavidin-gold beads to visualisethe extent of biotinylation.

[0296] a. Preparation of phage.

[0297] An oestradiol-binding phage (MT31C) was grown from a bacterialglycerol stock in 50 ml 2TY/2% glucose/1 μg/ml ampicillin (2TYGA) for 6hours at 37° C. M13KO7 helper phage (Stratagene) was added to theculture to a multiplicity of infection (moi) of approximately 10(assuming that an OD 600 mm of 1 is equivalent to 5×10⁸ cells per ml ofculture). The culture was incubated stationary at 37° C. for 15 minutesfollowed by 45 minutes with light aeration (200rpm) at the sametemperature. The culture was centrifuged and the supernatant drainedfrom the cell pellet. The cells were resuspended in 50 ml 2TYAK (2TYmedia supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin),and the culture incubated overnight at 30° C. with good aeration (300rpm). Phage particles were purified and concentrated by two polyethyleneglycol (PEG) precipitations (Sambrook, J., Fritsch, E. F., and Maniatis,T. (1990). Molecular Cloning—A Laboratory Manual. Cold Spring Harbour,N.Y.) and resuspended in PBS to 10¹² transducing units (tu)/ml.

[0298] b. Biotinylation of phase.

[0299] Phage were diluted to an approximate concentration of 2×10¹⁰ perml in a total volume of 500 μl. 5 μl of mouse Mab directed agains thegene 3 protein were then added to the phage and incubated at roomtemperature for 1 hour. 5 ml of an anti-mouse-IgG-HRP conjugate (Sigma)were then added to the phage and incubated at room temperature for afurther 1 hour. The phage were then treated with biotin tyramine byadding 50 μl of 1M Tris-HCl pH 7.4 to the phage mix, followed by 4 μl ofbiotin tyramine stock solution and 2 μl of hydrogen peroxide (Sigma).The reaction was allowed to proceed at room temperature for 10 minutes,and the biotinylated phage then stored at 4° C. overnight.

[0300] c. Streptavidin-gold labelling of biotinylated phage.

[0301] EM grids were blocked in 0.1% gelatin and phage samples thenapplied. The phage were then labelled with streptavidin-5 nm colloidalgold (Sigma) at an approximate concentration of 2×10¹¹ particles per ml.A number of images of the biotinylated ends of phages were generated.When the anti-gene 3 antibody was omitted no gold labelling of the phageends could be observed.

[0302] d. Estimation of the distance over which biotinylation hasoccurred using this system.

[0303] The number of gold particles found to localise to individual endsof phages in the electron micrographs were counted and the data wereused to generate a distribution histogram (FIG. 2). Data from twoseparate labelling experiments were pooled to generate the histogram.Theaverage number of gold particles associated with the phage ends wasfound to be 6.6, giving an average radius of biotinylation of 7.2 nm.Using this method the biotinylation range observed was from 5 nm to 25nm, 5 nm being the limit of resolution of the experiment. A typicalglobular protein has a diameter of around 4 nm, hence the biotinylationrange is of the order of 1 to 5 protein diameters.

[0304] e. Adjusting the distance of biotinylation.

[0305] To increase the distance over which biotinylation occursHRP-conjugated molecules of various lengths may be used. For example, aphage antibody with a specific binding characteristic may be HRPlabelled and then used to guide the biotinylation of phage antibodiesfrom the library. A phage particle is normally around 1 μm long, hencethis would give a radius of biotinylation of 10 nm to 1 μm. Similarlyother molecules of shorter or longer lengths may be used e.g.streptavidin-dextran-HRP conjugates, or beads of defined sizes such asMACS beads (Miltenyi Biotec), which have a diameter of 50 nm and can becoupled either directly, or indirectly via biotin-streptavidin, to HRP.Iterations of the biotin tyramine reaction may be performed to broadenthe area over which biotinylation is occurring. Example 10 describes avariation on the signal transfer technique which uses a lightactivatible streptavidin molecule with a short spacer arm (18 Angstrom).This procedure will only allow signal transfer to molecules bindingimmediately adjacent to the guide molecule.

EXAMPLE 13 STEP-BACK SELECTION TO ISOLATE PAGE ANTIBODIES WHICH INHIBITLIGAND BINDING.

[0306] This example describes use of the biotin tyramine signal transferselection procedure in a two step manner to isolate antibodies whichinhibit binding of the initial marker ligand to cells. This proceduremay be applied to the generation of inhibitors to any ligand, smallmolecule, or antibody. The process as exemplified here involves aninitial first stage of the selection to biotinylate and capture phageantibodies which bind around the site of ligand binding. Thebiotinylated phage are then used directly (with no need foramplification) to guide a second stage of selection using cells in theabsence of ligand. In this way antibodies which bind in the ligandbinding site can be biotinylated by signal transfer procedure, thencaptured and screened for inhibition of ligand binding. Such a scheme isoutlined in FIG. 3. The example described here uses phage to direct thesecond stage selection, but scFv may also be used either as a populationof scFv molecules, or by individual clone isolation and purification, asmay any other suitable binding molecule such as an antibody or bindingfragments thereof. The system used in this example is the same as thatdescribed in Example 9. MIP-1α was used as the guide ligand on purifiedCD4+ lymphocytes.

[0307] a. First stage selection

[0308] CD4+ cells were purified from blood as described in Example 9part a. The first stage selection procedure was then carried out exactlyas described in Example 9 part b), except that phage were captured on 90μl preblocked streptavidin-coated Dynal beads. After washing the beadswere resuspended in 90 μl PBS and 30 μl removed to titre the phagepresent on the beads. The remaining 60 μl were again taken out ofsolution using the magnet and phage were eluted from the beads using 100μl 100mM triethlyamine for 10 minutes at 37° C., and then neutralisedwith 50 μl 1M Tris-HCl pH 7.4. After elution the beads were taken out ofsolution and the supernatant containing the biotinylated phage was takenfor use in step two. The remaining beads were retained and used toinfect E coli TG1 to ascertain the phage titre remaining on the beadsafter elution.

[0309] The following titres were obtained: Total number of phagecaptured on the 1.7 × 10⁴ Dynal beads: Total number of phage retained onthe 2.2 × 10³ beads after TEA elution: Therefore total number eluted:1.5 × 10⁴

[0310] b. Second stage selection

[0311] The population of biotinylated phage which had been recoveredfrom the first stage of the selection was added directly to 1×10⁶ CD4+lymphocytes in a total volume of 200 μl in MPBS. Phage were allowed tobind to the cells for 1 hr at room temperature, and cells were thenwashed 3 times in 1 ml PBS. Cells were pelleted at 4000 rpm for 2 min ina minifuge between washes. A further aliquot of the scFv phage library(2×10¹² phage) was then added to the cells in 1 ml MPBS and allowed tobind for 1 hr at room temperature. Cells were washed 3 times in PBS asabove and then resuspended in 200 μl MPBS containing 2 μl ofstreptavidin-HRP complex (Amersham). This was allowed to bind for 30 minat room temperature, and cells were then washed as before. Biotintyramine treatment of the cells was carried out as described in Example9 part b). Cells were then lysed by resuspension in 100 μl PBST and 30μl preblocked streptavidin-coated Dynal beads were added to the lysate.Beads and lysate were rotated at room temperature for 20 min, and thebeads then taken out of solution on a magnet. Beads were washed 3 timesin 1 ml PBST, followed by 3 times in 1 ml PBS. Washed beads were used todirectly infect an exponentially growing culture of E coli TG1. A totalof around 4×10³ clones were recovered from this selection procedure.

[0312] c. Growth of single selected clones for immunoassay.

[0313] Individual colonies from the second step of the selectionprocedure were used to inoculate 100 μl of 2TYGA into individual wellsof tissue culture plates. Plates were incubated at 30° C. overnight withmoderate shaking (200rpm). Glycerol to 15% was added to each well andthese master plates stored at −70 C. until ready for analysis.

[0314] d. Phage ELISA to identify anti-CD4+ scFv's.

[0315] Cells from the master plate were used to inoculate fresh 96 welltissue culture plates containing 100 μl 2TYGA per well. These plateswere incubated at 37° C. for 6-8 hr. M13KO7 was added to each well to anmoi of 10 and incubated stationary for 30 min then 30 min with gentleshaking (100rpm), both at 37° C. The plates were centrifuged at 2000rpmfor 10 min and the supernatant removed. Each cell pellet was resuspendedin 100 μl 2TYAK and incubated at 30° C. overnight. Each plate wascentrifuged at 2000 rpm for 10 min and the 100 μl of supernatant wasrecovered and blocked in 20 μl 18% M6PBS (18% skimmed milk powder,6×PBS), stationary at room temperature for 1 hr.

[0316] CD4+ cells were isolated as described (Example 9, part a) and1×10⁵ cells were spun onto 96 well culture wells which had beenprecoated with poly-L-lysine for 30 min at room temperature. Cells wereblocked in 100 μl MPBS for 2 hours at 37° C., and rinsed once in PBS.The phage supernatants were then added to the cells and incubated for 1hr at room temperature, then washed 3 times in PBS. 100 μl of a 1:5000dilution of sheep anti-fd antibody (Pharmacia) in MPBS was added and theplates incubated at room temperaure for 1 hr. Plates were washed 3 timesin PBS and 100 μl of a 1:5000 dilution of donkey anti-sheep alkalinephosphatase conjugate (Sigma) in MPBS was added and incubated for 1 hrat room temperature. Plates were washed 3 times in PBS and alkalinephosphatase activity was visualised using the chromagenic substrate pNPP(Sigma). Absorbance was measured at 405 nm using a microtitre platereader. 45 individual colonies were assessed for CD4 cell binding inthis way, and 25 were found to be positive. 6 of these were taken atrandom for further analysis.

[0317] e. Assessment of anti-CD4 scFv's to inhibit binding of MIP-1α toCD4+ cells.

[0318] 6 scFv's were purified using nickel agarose metal affinitychromatography (Quiagen). 1×10⁵ CD4 cells were preincubated with thepurified CD4+-binding scFv's, or with an irrelevant control scFv for 1hr at room temperature in PBS containing 0.1% BSA in a total volume of100 μl. Approximately 5-10 μg of scFv was used per sample Cells werepelleted at 4000 rpm in a minifuge and washed once in 1 ml PBS.Biotinylated MIP-1α (R and D Systems) was made up according tomanufacture's instructions 5 μl (equivalent to 5ng) added to the cellsin 100 μl MPBS and incubated at room temperature for I hr. Cells werewashed as before. 100 μl of streptavidin-FITC (Sigma) at a dilution of1:100 in MPBS was added and incubated for 30 min at room temperature,and cells were washed as before. Fluorescence was detected using aCoulter Epics-XL flow cytometer. MIP-1α gave significant shift in thefluorescence of the cells when no scFv, or control scFv was added to thecells. In the presence of scFv from the selected clones MIP-1α bindingto the cells was significantly inhibited. Inhibition varied from cloneto clone.

EXAMPLE 14 BIOTIN TYRAMINE SELECTION IN SOLUTION USING A PEPTIDE PHAGELIBRARY

[0319] 9E10 is a commercially available mouse monoclonal antibody whichrecognises a peptide which is part of the cellular myc protein (Munro,S. and Pelham, H. R. B. (1986), Cell 46, 291-300). This experiment wasdesigned to select for peptides from a large peptide library which bind9E10. 9E10 was conjugated to HRP to allow biotin tyramine-directedselection in solution. This can be considered as a novel method ofepitope mapping antibodies, or other protein binding domains.

[0320] a. Construction of the peptide library

[0321] In this example, the peptide library used was constructed asdescribed by Fisch et al (I. Fisch et al (1996) Proc. Natl. Acad. Sci.USA 93 7791-7766) to give a phage display library of 1×10¹³ independentclones.

[0322] b. Conjugation of the anti-myc antibody (9E10) to HRP

[0323] 1 ml of 1 mg/ml 9E10 IgG was conjugated to HRP using the PierceEZ-link malemide activated HRP kit (cat no. 31494). 1 ml of 9E10 wasadded to the vial containing 6 mg of 2-mercaptoethylamine (MEA) in 100μl conjugation buffer. This was incubated for 90 min at 37° C. Thesolution was allowed to cool to room temperature and the MEA wasseparated from the reduced IgG using the desalting column. The columnwas pre-equilibrated by washing with 30 ml of maleimide conjugationbuffer and the 1.1 ml of IgG/MEA solution was applied to the column. Theconjugate was eluted using the maleimide conjugation buffer. 1 mlfractions were collected and fractions 5 and 6 were found to contain themajority of the protein. Fractions 7 and 8 contained smaller amounts ofprotein and were retained for control selections. Fractions 5 and 6 werepooled and 1 mg of maleimide activated HRP was added to the IgG andallowed to react for 1 hour at room temperature.

[0324] c. First round selections using 9E10-HRP and the peptide phagedisplay library

[0325] Approximately 1×10¹³ phage were used per selection. 6 μg of9E10-HRP conjugate were added to the peptide phage in a total volume of1 ml PBS with 2% marvel (MPBS). Control selections were also carried outusing 6 μg of unconjugated 9E10. All selections were carried out in 1ml. Phage and antibody were allowed to bind overnight at 4° C. 50 μl 1MTris-HCl pH 7.4, 4 μl biotin tyramine and 2 μl hydrogen peroxide werethen added to the selection and allowed to react for 10 min at roomtemperature. 100 μl of streptavidin-coated magentic beads (Dynal) whichhad been preblocked for 30 min in MPBS were then added to the selectionand rotated at room temperature for 30 min. Magnetic beads were thenbrought out of solution using a magnet and washed with 3×1 ml of PBScontaining 0.It Tween, followed by washing with 3×1 ml of PBS. The beadswere resuspended in a final volume of 100 μl PBS and used to directlyinfect 5 ml of an exponentially growing culture of E.coli TG1. Infectionwas carried out by incubation stationary at 37° C. for 30 min, followedby 30 min slow shaking (200rpm) at 37° C. Phage were plated out on 2TYmedium containing 100 μg/ml tetracyclin (2TYT). Colony counts gave thephage titre.

[0326] d. Second and third round selections using 9E10-HRP and thepeptide library.

[0327] The plates were scraped into 5 ml of 2TY. 50 μl of this platescrape was then added to 50 ml of 2TYT and grown overnight at 30° C.with aeration. 1 ml of the resultant cell suspension was pelleted at6000 rpm in a minifuge and 100 μl of 10×MPBS added to the supernatant.9E10-HRP conjugate, unconjugated were then added to the blocked phage asselections carried out exactly as the first round described in part c.above. The selection was repeated so that a total of three rounds ofselection were performed. The number of phage recovered in the outputpopulations at each round was as follows: 9E10-HRP 9E10 Round 1 5.2 ×10⁵ 2.0 × 10⁴ Round 2 1.2 × 10⁶ 5.9 × 10⁴ Round 3 8.0 × 10⁵ 2.6 × 10⁵

[0328] e. Screening the output populations for binding to 9E10

[0329] Individual colonies from the third round of selection were usedto inoculate 96 well tissue culture plates containing 100 μl of 2=YT perwell and clones were grown overnight at 30° C. with good aeration(300rpm). Plates were centrifuged at 2000 rpm and the 100 μl fromsupernatant from each well was recovered and blocked in 20 μl 18% M6PBS(18% milk powder, 6×PBS) stationary at room temperature for 1 hour.ELISA plates which had been blocked overnight at 4° C. with 50 μl of 10μg/ml 9E10, or 50 μl PBS alone were washed in PBS and then blocked for 2hours stationary at 37° C. in 3MPBS. ELISA plates were washed in PBS andthe blocked phage supernatants then added to the ELISA plate. The plateswere incubated stationary at room temperature, then washed three timeswith PBST, followed by three washes with PBS. 50 μl of anti-gene 8-HRPconjugate diluted at 1:5000 in 3MPBS were then added to each well andthe plates incubate at room temperature for 1 hour. Plates were washedas before and the ELISA developed for 1 hour at room temperature with 50μl of TMB substrate. Development was stopped by the addition of 25 μl of1M H₂SO₄.

[0330] f. Results of the screening

[0331] 95 clones from the third round of selection using the 9E10-HRPconjugate, and 95 from the unconjugated 9E10 selection were screened byELISA. 3 positives were identified as binding 9E10, but not PBS coatedplates from the 9E10-HRP selection, whereas no positives were found fromthe control unconjugated 9E10 selection. The three positive clones wererechecked by ELISA as above on an unrelated mouse monoclonal antibodyand did not give any signal, demonstrating that they bind specificallyto the 9E10 Mab.

[0332] g. Sequencing 9E10-binding clones

[0333] Clones found to be positive for binding to 9E10 were analysed byDNA sequencing as described by Fisch et al. All three clones were, foundto be identical. None had a peptide insert in Exon 1, and all a 10 aminoacid peptide sequence inserted in Exon 2 which had some homology to themyc tag, as shown below:

[0334] Selected sequence: P M P H A E G K S T

[0335] Myc tag: G A A E Q K L I S E E D L M

[0336] In summary 9E10-specific clones have been identified from thepeptide library, which have some homology to the myc tag. Thisdemonstrates that biotin tyramine selections can be successfully carriedout in solution, and can be carried out on non-antibody libraries.

EXAMPLE 15 CHARACTERISATION OF CONES WHICH BIND TO CD4+ CELLS, BUT NOTTO THE CHEMOKINE RECEPTOR CC-CKR5, BY WESTERN BLOTTING AND ICC.

[0337] Example 9 described the selection of phage antibodies which bindto a chemokine receptor. Phage selections were carried out on CD4+ cellsusing biotinylated MIP-1α, followed by streptavidin-HRP to guide theselection. 30/95 phage selected in the presence of the biotin tyramineand MIP-1α recognised CD4+ lymphocytes. 13 of these clones were found tobe positive for the CC-CKR5 chemokine receptor for which MIP-1α is aligand, leaving 17 clones which bind to CD4+ cells, but to anotherantigen to be discertained (Example 9 part c). These clones mayrecognise antigens which are normally found in close proximity to MIP-1αreceptors, or are MIP-1α receptors other than CC-CKR5 (CC-CKR1 andCC-CKR4 both bind to MIP-1α) . Identification of the antigens to some ofthese CD4+ binding clones allows examination of protein-proteininteractions on the cell surface, and exemplifies the potential ofbiotin tyramine selection as a tool for discovering novelprotein-protein interactions.

[0338] Three clones, CD4A2, CD4E1 and CD4D2 were chosen at random fromthe 17 CD4+ binding clones and were subjected to further analysis toidentify their antigen partners. Initial studies involved probingwestern blots of membrane fractions prepared from CD4+ cells withpurified scFv from the 3 clones. Immunocytochemistry on CD4+ cells wasalso carried out using the scFv's.

[0339] a. Preparation of CD4+ cell membrane fractions.

[0340] CD4+ lymphocytes were prepared as described in Example 9, part(a). Membrane preparations were then generated as follows. Approximately1×10⁶ cells were resuspended in 1 ml of 12mM Tris-HCl, pH 7.5 in 250mMsucrose. Cells were lysed by three cycles of freeze thawing, and thelysates were homogenised in a ground glass homogeniser. The homogenatewas centrifuged at 270×g for 10 min at 4° C. to pellet the nuclearfraction. The supernatant was then centrifuged at 8000×g for 10 min at4° C. to pellet the mitochondrial and lysosomal fractions. The finalcentrifugation to pellet the plasma membrane fraction was carried out at100 000×g for 60 min at 4° C., and the membrane fractions wereresuspended in 100 μl PBS and stored at −70°C.

[0341] b. Western blotting of membrane fractions.

[0342] 4-20% Novex gradients were run under non-reducing, denaturingconditions at 125V for 1.5 hr, and blotted in at 25V for 1.5 hr.Blotting was carried out in the Novex apparatus exactly as recommendedby the manufacturers using Hybond-C membrane (Amersham).

[0343] c. Probing western blots.

[0344] Membranes were blocked for 45 min in MPBS and probed with 50 μgpurified scFv in 5 ml MPBS for 1 hr at room temperature. Blots werewashed in three changes of PBST, followed by three changes of PBS. 9E10at a 1:100 dilution in MPBS was then incubated on the membrane for 1 hrat room temperature. Washing was carried out as before, andanti-mouse-IgG-HRP antibody then added at a dilution of 1:5000 in MPBS.Blots were developed using ECL substrate (Amersham) and exposed toautoradiographic film.

[0345] Clone CD4E1 gave a band of approximately 29 kDa

[0346] Clone CD4D2 gave a band of approximately 31 kDa

[0347] Clone CD4A2 failed to give a specific band under denaturing gelconditions.

[0348] d. Immunocytochemistry (ICC) using scFv's on CD4+ cells.

[0349] Approximately 1×10⁵ CD4+ cells were spun onto poly-L-lysinesubbed slides using a Cytospin (Serotech). Slides were blocked in MPBSfor 2 hr at room temperature and a 1:10 dilution of the scFv in MPBSthen incubated on the slides for 1 hr at room temperature. Slides werewashed in PBS and detection was achieved using 1:100 dilution of 9E10,followed by a 1:500 dilution of anti-mouse-HRP, both diluted in MPBS andincubated for 1 hr at room temperature, with washing in PBS betweenincubations. CD4E1 and CD4D2 both gave clear staining of the cellmembranes

EXAMPLE 16 DEMONSTRATION OF THE USE OF SIGNAL TRANSFER SELECTION TOIDENTIFY NOVEL PROTEIN-PROTEIN INTERACTIONS.

[0350] To definitively identify the antigens which clones CD4A2, CD4E1and CD4D2 recognise, a lambda gtll cDNA expression library wasconstructed from mRNA from purified CD4+ cells and screened withpurified ScFv+s.

[0351] a. Isolation of messenger RNA

[0352] Messenger RNA was purified from a population of CD4 purifiedcells using a QuickPrep Micro mRNA purification kit (Pharmacia). ThemRNA was purified following manufacturer's instructions. The methodinvolved lysis of the cells in a buffered aqueous solution containingguanidinium thiocyanate and N-lauroyl sarcosine, the extract was thendiluted three fold with an elution buffer which reduces the guanadiniumconcentration to a level which is low enough to allow efficient hydrogenbonding between poly(A) tracts on the mRNA and the oligo(dT) attached tocellulose, but high enough to maintain complete inhibition of RNAses.The dilution step causes a number of proteins to precipitate, giving aninitial purification. The extract was clarified by short centrifugationat top speed in a minifuge and the supernatant transferred to amicrocentrifuge tube containing Oligo(dT)-cellulose. After 10 min,during which time the poly (A)+RNA binds to the oligo (dT)-cellulose,the tube was centrifuged at high speed for 10 sec, and the supernatantwas aspirated off the pelleted oligo (dT)-cellulose. Pelleted materialwas washed sequentially with 1 ml aliquots of high salt buffer and lowsalt buffer, each wash being accomplished by a process of resuspensionand brief centrifugation. After the last wash the pelleted material wasresuspended in 50 μl of low salt buffer and transferred to a MicroSpincolumn placed in a microcentrifuge tube, and the column was washed threetimes with 0.5 ml of low salt buffer. Finally, the polyadenylatedmateriel was eluted with prewarmed elution buffer (10mM Tris-HCl (pH7.5), 1 mM EDTA). The mRNA was precipitated by addition of a glycogencarrier, potassium acetate and ethanol. After precipitation the mRNA wasrecovered by centrifugation and resuspended in DEPC treated water.

[0353] b. cDNA Synthesis

[0354] cDNA was synthesised from the CD4 mRNA using a cDNA synthesis kitsupplied by Amersham International. The detailed protocol booklet wasfollowed. The 1st strand synthesis reaction contained hexamer primersand reverse transcriptase with mRNA as template. Second strand synthesiswas carried out with Ribonuclease H and DNA polymerase, and aftersynthesis the ends of the cDNA were made blunt by treatment with T4 DNApolymerase. The cDNA was then purified by phenol/chloroform extraction.

[0355] c. Construction of cDNA library

[0356] cDNA was cloned into the lambda gtll expression vector usingAmersham's cDNA rapid adaptor ligation module (RPN 1712) and the cDNArapid cloning module—gtll (RPN1714). Adaptors were added to the cDNA togive EcoR1 restriction cohesive ends, and cDNA with adaptors wereseparated from free adaptors by a column step.The adapted cDNA was thenligated into lambda gtll vector then packaged using an in vitropackaging kit. Resultant reactions were titred to access the librarysize, which was found to be 7×10⁵.

[0357] d. Screening the cDNA expression library with scFv

[0358] For immunoscreening host cells (Y1090) were infected with phagefrom the library and plated out on L top agarose. After 3.5 hours growthat 42° C., the plates were overlaid with nitrocellulose filterssaturated with 10 mM IPTG, an inducer of Lac Z gene expression, andincubated for a further 3.5 hours at 37° C. During this time, theplaques are transferred to the filter along with the β-galactosidasefusion proteins, released from the lytically infected cells. The filterswere carefully removed and washed briefly in PBS and then blocked inMPBST. Detection of positives was by sequential incubations with scFv ofinterest at a concentration of 10 μg/ml in MPBS, followed by 9E10 (1:100in MPBS) and then an anti-mouse HRP congugate (1:1000 in MPBS). Thefilters were washed between incubations in 3 changes of PBST. Signal wasdetected using an Enhanced Chemiluminescent system (Amersham ECL Kit).Plaques which were found to be positive from the first round ofscreening were picked and re-infected into a fresh culture of Y1090, andthe screening process repeated. This was carried out to ensure thereproducibility of the positive signal and to obtain clonal plaques.

[0359] e.Sequencing inserts from positive plaques.

[0360] Single plaques were picked into 100 μl SM buffer and left at 4°C. overnight. 5 μl of the eluted plaques was then taken and used astemplate for a standard 50 μl PCR reaction (0.5 μl TAQ Polymerase, 4 μl10mM dNTP, 5 μl 10×PCR buffer, 2.5 μl of each primer (10 μM), made up to50 μl with water). Primers used for sequencing were:

[0361] gtll screen5 5′ GAC TCC TGG AGC CCG

[0362] gtll screen3 3′ GGT AGC GAC CGG CGC

[0363] PCR products were then cleaned up and used in sequencingreactions as described previously (Example 2 part e), except that gtllscreen5, and gtll screen3 were used as sequencing primers. Resultantnucleotide sequences were then aligned to the NCBI data base using theBLAST programme (Altschul et al., J. Mol. Biol. (1990) 215, 403-410.).

[0364] f. Results of the sequence alignments lambda gt11 Degree of scFvclone clone Homology identity CD4E1 2.1.1 Rat CL-6  80% CD4A2 3.1.1TRIP-4 (human) 100% CD4D2 10.1.1  26S proteosome p31 (human) 100%

[0365] CD4e1

[0366] This was found to recognise a lambda clone containing an insertwhich had homology to a rat protein called CL-6, which is aninsulin-induced growth response protein. This protein is a proteintyrosine phosphatase (PTP). PTP's are a family of intracellular andintegral membrane phosphatases which dephosphorylate tyrosine residuesin proteins. PTP's have been implicated in the control of normal andneoplastic growth and proliferation. PTPs have also been implicated inT-cell signal transduction pathways, where they are involved in couplingreceptors to the generation of second messenger inositol triphosphate.The DNA fragment isolated here has 80% identity at the nucleotide levelwith the rat CL-6 protein, and hence is probably the human homologue.CL-6 is an approximately 30kDa protein.

[0367] The rat gene CL-6 was identified by R. F. Diamond et al. (1993,Journal of Biological Chemistry 268, 15185-15192) as a gene which wasinduced in regenerating liver and insulin-treated Reuber H35 cells, arat hepatoma cell line which grows in response to physiologicalconcentration of insulin and retains some properties of regeneratingliver. CL-6 was one of a panel of 41 novel growth response genesidentified in this study, and was found to be the most abundantinsulin-induced gene. CL-6 is induced as an immediate-early gene in theliver cells, and its immediate-early induction during liver regenerationsuggests that it is regulated by early stimuli, and not by insulinalone. CL-5 mRNA expression was found to be highest in liver and kidney,but showed some expression in most tissues. The CL-6 protein ispredicted to be highly hydrophobic, and may be a membrane-associatedprotein. CL-6 is likely to have a role in the tissue-specific aspects ofcellular growth, involved in the maintenance of normal liverarchitecture or metabolism during regeneration and foetal development.

[0368] CD4A2

[0369] This clone was found to recognise thyroid receptor interactingprotein 4 (TRIP4). Thyroid hormone receptors (TRs) are hormone-dependenttranscription factors that regulate expression of a variety of specifictarget genes. Thyroid interacting proteins are thought to play a role inmediating the TR's response to hormone binding.

[0370] CD4D2

[0371] This clone was found to recognise the p31 (31 kDa) subunit of thehuman 26S proteosome. Proteosomes are involved in theubiquitin-dependent proteolytic pathway and in antigen processing, andthere is evidence that they are found in close proxmity to, orassociated with the plasma membranes in vivo.

[0372] g. Summary of results

[0373] It has been demonstrated that the signal transfer selectionprocedure can be used to select for antibodies, or other bindingspecies, which bind to antigens found in the vicinity of the originaltarget antigen, but which do not recognise the target antigen itself.CD4A2, CD4E1 and CD4D2 are three examples of this. The antigens whichthese three antibodies recognise have been identified by screening acDNA expression library. The antigens identified by cDNA screening fitwith the predicted sizes of the antigens which CD4 E1 and CD4D2 bind toon western blotting i.e. the human homology of CL-6 (30kDa), the p31subunit of the 26S proteosome (31 kDa).CDA2 recognises the TRIP4protein, which has an estimated molecular weight of 32 kDa. Theantibodies stain CD4+ cell membranes by ICC, as does MIP-1α, the ligandfor the CC-CKR5 receptor which was used to guide the signal transferselection. Hence signal transfer selection has been used to identify apanel of antigens which are found in close proximity (probably up to 25nm) to MIP-1α receptors on the surface of CD4+ cells. This is ademonstration of the use of signal transfer selection as a means ofidentifying novel protein-protein interactions, and to identify novelgenes. The CL-6 gene has previously only been identified in rat, andsignal transfer selection has enabled the cloning of the human homologueof CL-6.

EXAMPLE 17 BIOTINYLATION OF CD4E1 PHAGE ON THE CELL SURFACE USING MIP-1αTO DIRECT THE BIOTINYLATION

[0374] Clone CD4El has been selected by virtue of the fact that it bindsto an antigen found close to MIP-1 α binding sites on CD4+ cellsurfaces. It should therefore be possible to use biotinylated MIP-1αbound to streptavidin-HRP to catalyse biotin tyramine deposition ontoCD4E1 phage bound co the CD4+ cell surface to demonstrate that the CD4E1antigen is normally found in close association with MIP-1αreceptors.This was tested by incubating cells with biotinylated MIP-1α ,streptavidin-HRP and CD4E1 phage, treating with biotin tyramine and thenrecovering the biotinylated phage and titring. Recovery of phage usingthis system was compared to recovery when phage which bind at a site onthe CD4+ cell surface which is remote from the MIP-1α binding sites wereincubated with the cells, or when a biotinylated ligand (biotinylatedVCAM) which binds at another remote site on the CD4+ cell surface wasused in conduction with CD4E1 phage.

[0375] a.Biotinylation of phase on the cell surface.

[0376] CD4+ cells were purified as described in Example 9. CD4E1 phage,or phage from a CD4+ binding clone (CLA4) were prepared as described inExample 12. 1×10⁶ cells were incubated for 1 hr with 5 ng ofbiotinylated MIP-1α , or 5 ng of biotinylated VCAM in a total volume of100 μl PBS/BSA. Streptavidin-HRP (1:1000 dilution in PBS/BSA) was thenadded to the cells and incubated for 30 min. Cells were washed in PBS,and then 10¹¹ phage added in PBS/BSA and allowed to bind to the cellsfor 1 hr at room temperature. Cells were washed in PBS and then treatedwith biotin tyramine as described before. Cells were washed in PBS andthen lysed in PBS containing 0.1% Tween and biotinylated phage werecaptured on streptavidin-coated. Beads were washed three times in PBSTand three times in PBS, then infected directly into an exponentiallygrowing culture of E coli TG1.

[0377] b. Results of phage biotinylation.

[0378] The number of phage captured on beads was calulated from thetitres. If no biotinylation reaction was carried out approximately 10⁷CD4E1 and CLA4 phage were found to bind to the cell surface. PhageLigand Total Number of phage recovered CD4E1 MIP-1α 2000  CD4E1 VCAM 800CD4E1 — 400 CLA4 MIP-1α 600 CLA4 VCAM 800 CLA4 — 200

[0379] The number of phage recovered was at least 2.5 times higher whenCD4E1 phage was incubated with the MIP-1α, than the recovery attained inthe various control samples. This provides indication thatHRP-conjugated MIP-1α is able to specifically biotinylate CD4E1 phagebecause CD4El recognises an antigen which is found in close proximity(within 25 nm ) to the MIP-1α receptor.

EXAMPLE 18 USE OF BIOTIN TYRAMINE AS A SIGNAL AMPLIFICATION REAGENT INFLOW CYTOMETRY.

[0380] Signal transfer can also be used as an amplification system forenhancing fluoresence signals in flow cytometry. This is achieved byallowing a HRP-conjugated antibody, or ligand to bind to cells. Cellscan then be treated with hydrogen peroxide and biotin tyramine, asdescribed for the selection procedure. This will cause biotin tyraminedeposition around the antibody, or ligand binding site on the cellsurface. Streptavidin-fluorescein (FITC) can then be added to the cells.This will bind to the newly deposited biotin on the cell surface andgive an enhancement in signal as compared to a standard FITC celllabelling protocol using FITC-conjugated antibody or ligand. This hasbeen shown to be the case by comparing the labelling achieved onpurified CD4+ lymphocytes using either an anti-CD4+ antibody, followedby anti-mouse-FITC, or by using anti-mouse-HRP followed by biotintyramine treatment and then streptavidin-FITC.

[0381] a. Cell labelling.

[0382] CD4+ lymphocytes were purified as described in Example 9. Cellswere incubated with the anti-CD4+ antibody (Sigma), at a dilution of1:1000 in PBS/BSA. Cells were washed in PBS, and then either of the twosecond antibodies (anti-mouse-FITC, or anti-mouse-HRP) were added to thecells, at a dilution of 1:1000 in PBS/BSA. 1×10⁵ cells were used persample. Cells were washed in PBS and either detected directly(anti-mouse-FITC), or treated with biotin tyramine as describedpreviously. Biotin tyramine was added over a range of concentrationsfrom 0.25 μg/ml up to 100 μg/ml, in order to determine the concentrationat which the optimal signal enhancement occurred. After biotin tyraminetreatment cells were again washed in PBS, then streptavidin-FITC wasadded at a diltuion of 1:1000 in PBS. Cells were analysed by flowcytometry.

[0383] b. Flow Cytometry Results

[0384] The peak position (i.e. a measure of the fluorscence achieved)obtained using the different biotin tyramine concentrations was plotted.The results are shown in FIG. 4. As can be seen from the figure, optimalenhacement with biotin tyramine was obtaining using a concentration of12.5 μg/ml. The optimised peak position obtained using theanti-mouse-FITC second antibody was 60 fluorescence units, hence the useof biotin tyramine has efficiently enhanced this signal over 5-fold(from 60 to 330 fluorescence units).

EXAMPLE 19 ITERATION OF BIOTIN TYRAMINE TREAMENT TO GIVE FURTHER SIGNALENHANCEMENT

[0385] Repeated rounds of biotin tyramine treament may be carried outbefore a final detection step, using streptavidin-FITC. The repeatedrounds are achieved by an initial biotin tyramine treatment, followed bythe addition of streptavidin-HRP and then a further biotin tyraminetreatment. This example demonstrates the effective use of two rounds ofbiotin tyramine treatement to generate further signal enhancements. Amixed Ficoll purified cell preparation (containing monocytes,lymphocytes and granulocytes) and labelling with anti-CD36 antibody,which is a marker of monocytes, was used here as a model system.

[0386] a. Cell labelling.

[0387] Ficoll purified cells (1×10⁶ cells per sample) were incubatedwith anti-CD36 antibody for 30 min at room temperature, diluted (1:1000)in PBS/BSA. Cells were washed in PBS/BSA, and then incubated with ananti-mouse-HRP conjugate (1:1000 in PBS/BSA) for 30 min at roomtemperature. Cells were washed as before, and then treated with biotintyramine at 12.5 μg/ml . Samples which were to receive just one biotintyramine treatment were then washed and incubated with streptavidin-FITC(1:1000 in PBS/BSA). The samples which received a further treament ofbiotin tyramine were incubated with streptavidin-HRP (1:1000 in PBS/BSA)for 30 min at room temperature, then washed and treated with biotintyramine as before. Cells were washed again, and then incubated withstreptavidin-FITC as before.

[0388] b. Results

[0389] Samples were analysed by flow cytometry and the fluorescenceshifts overlayed, as shown in FIG. 5. As can be seen from the figureiteration of the biotin tyramine treament results in a 2.5 fold shift inthe average fluorescence level of the cells. This is to expected giventhe observation that biotinylation may occurs over a range of up to 25nm from the original site of HRP localisation. Assuming the first biotintyramine treatment biotinylates proteins in a circle of radius 25 nmfrom the HRP, then this would give an area of biotinylation of π25² nm²,which is 1963 nm². In the case of the second treatment with biotintyramine the biotin desposited in this area is then saturated withstreptavidin-HRP, so blocking binding of any streptavidin-FITC. Thebiotin tyramine treament is repeated giving a further area ofbiotinylation of π50²-π25², which is 5887 nm², which is an area threetimes the size of the original circle of biotin deposition. This fitswith the experimentally observed observed fluorscence shift of around2.5 fold.

[0390] The fluorescence shift observed after iterations of biotintyramine treatment may be used to assess cellular copy numbers of cellsurface proteins. If a protein is rare on a cell surface then thefluorescence signal should carry on increasing with successive rounds ofbiotin tyramine treatment until the cell surface is saturated. If aprotein is expressed at high copy number on a cell surface thefluorescence signal will saturate sooner because the circles ofbiotinylation will overlap.

EXAMPLE 20 USE OF BIOTIN TYRAMINE TO SPECIFICALLY BIOTINYLATESUBPOPULATIONS OF CELLS TO ALLOW THEIR SUBSEQUENT PURIFICATION

[0391] This example demonstrates using biotin tyramine to specificallybiotinylate subpopulations of cells within a complex mix and then tocapture the biotinylated cells to give an enriched population. Thesystem chosen here uses an anti-CD36 mouse monoclonal antibody(Immunotech) which is a monocyte cell surface marker. A mixture ofmonocytes, lymphocytes and granulocytes was purified from blood on aFicoll density gradient. Lymphocytes and granulocytes do not expressCD36, hence the antibody should specifically biotinylate monocytes. Thetechnique is equally applicable to any molecule which binds cellsurfaces, and to any cell type, virus particle, bead or other populationof particles displaying an sbp member or epitope.

[0392] a. Purification of cells from buffy coat.

[0393] Adult buffy coat blood from Cambridge Blood Transfusion Servicewas diluted 1:2 with Dulbeccos PBS (Tissue culture grade) then loadedonto 1077 density Ficoll Hypaque (Sigma). This was then spun at 1500 rpmfor 30 min at room temperature with brake off. Cells at the interfacewere removed and washed once with PBS. Red cells were removed by using awhole blood erythrocyte lysing kit from R&D systems (Cat. no. WL1000).Cells were resuspended in 5 ml of lysing reagent and left for 5 min atroom temperature then spun at 1000 rpm for 5 min and washed in 10 ml ofwash reagent and again spun at 1000 rpm for 5 min. Cells wereresuspended in PBS/0.5% BSA/2mM EDTA (PBE) and then counted. In each ofthe following experiments 2.4×10⁶ cells were used.

[0394] b. Antibody incubations and biotin tyramine treatment

[0395] Cells (2.4×10⁶) were incubated with mouse IgG₁ anti human CD36antibody (Immunotech 0765) (2 μg/10⁵ cells) for 30 minutes at 4-8° C.,washed in PBE and spun at 1000 rpm for 5 min. Incubation with goatanti-mouse HRP conjugated antibody (1:1000 dilution) was the same as forthe anti CD36 antibody. All antibodies were diluted in PBE. Cell pelletswere resuspended in 100 μl of 50 mM Tris-HCl pH 7.4 with 2 μlbiotin-tyramine (5 μg) and 1 μl of H₂O₂. This was left at roomtemperature for 10 minutes and then washed with 5 ml of PBE.

[0396] c. Capture of biotinylated cells

[0397] This was carried out using streptavidin MACS beads (MiltenyiBiotec) as per manufacturer's instructions. Cells from the previoustreatment was resuspended in 80 μl PBE with 20 μl of MACS streptavidin(Cat no. 481-01). Incubation was for 15 min at 4° C. Cells were washedin PBE, resuspended in 100 μl of PBE and loaded onto a MACS columnenclosed in a MACS magnet. Cells were allowed to run in to the column,and then the column was washed with 2×1 ml of PBE to remove unboundcells. Cells were eluted from the column by removing the column from themagnet, adding 1 ml of PBE, and then pushing the plunger into thereservoir to push the PBE through the column. Cells were eluted into aneppendorf and then spun at 4000 rpm for 5 min in a microcentrifuge. Cellpellets were resuspended in 80 μl of PBE and 20 μl of anti-CD36 antibodyconjugated to fluorescein (Immunotech 0766). Cells were incubated in thedark at 4° C. for 20 minutes. Samples were then analysed by flowcytometry.

[0398] d. Results

[0399] Anti-CD36 antibody, followed by anti-mouse-HRP and biotintyramine treatment was successful in biotinylating a subpopulation ofcells which were subsequently captured on streptavidin beads. Thecaptured cells were found to be CD36 positive and were at theappropriate position by forward and side scatter in the flow analysis tobe monocytes (FIG. 9).

EXAMPLE 21 BIOTINYLATION OF PHAGE PARTICLES IN SOLUTION TO VALIDATEBIOTIN-TYRAMINE PREPARATIONS.

[0400] A phage preparation was made as described in Example 12 part a).Phage particles were diluted to a titre of 1×10⁹ phage in 1 ml and 1 μlof a HRP-conjugated mouse Mab recognising the gene 8 protein HRPconjugate (Pharmacia) was added to the phage in solution. This wasincubated at room temperature for 1 hr, and the phage were then treatedwith biotin tyramine, as described in Example 12 part b). Additionaldilutions of biotin tyramine ranging from a 1:100 dilution of the normalstock solution, up to 100 fold excess over the normal concentrationsremained constant. Biotinylated phage were then captured on 30 μlpreblocked streptavidin-coated beads and the beads washed as describedbefore. Phage captured on beads were titred and the optimal biotintyramine concentration which gave maximal biotinylation was established.The results are shown in FIG. 7.

[0401] This provides a means of validating preparations of biotintyramine, and allows comparison between different batches. The optimalbiotin tyramine was evaluated for two different preparations of biotintyramine, and was found to be comparable. TABLE 1 No. No. 2nd phagephage % Mab in TEA recovered Eluate Expt Phage 1st (HRP) eluate on beadsre- No. type Mab (1/2500) BT (× 10⁵) (× 10³) covered (i) CEA6 1/100 + +5.6 4400 0.80 (ii) CEA6 1/1000 + + 3.9 1000 0.25 (iii) CEA6 1/10000 + +9.1 1500 0.16 (iv) CEA6 1/100 + − 5.3  280 0.05 (v) CEA6 1/100 − + 4.2 840 0.20 (vi) CEA6 − + + 4.6  440 0.10 (vii) OPI 1/100 + + 1.8  80 0.04

[0402] TABLE 2 Round 1 No. phage No. phage Selection phage in eluaterecovered on % Eluate round No. taken 1st Mab (× 10⁵) beads (× 10²)recovered 1A 1/100 7.7 >300 >4 1B − 3.4 >200 >6 2 1A 1/100 1.8 4.13 0.232 1A 1/1000 1.4 3.64 0.26 2 1B − 2.8 1.11 0.04 2 1A − 1.5 1.87 0.12

[0403] TABLE 3 Round 1 No. Clones Selection phage taken screened CEA +ve % CEA + ve 1A — 94 3 4 1B — 94 0 0 2A 1A 48 13  27  2B 1A 65 11  17 2C 1A 48 4 8 2D 1B 25 1 4

[0404] TABLE 4 Clone Ic_(off) (s⁻¹) SS1A4 8.9 × 10⁻² SS1A11 7.2 × 10⁻²SS1G12 3.3 × 10⁻² SS22A8 7.8 × 10⁻² SS22B7 1.9 × 10⁻² SS22B1 1.3 × 10⁻²SS22D12 3.4 × 10⁻² SS22E4 7.5 × 10⁻³ SS21B7 2.0 × 10⁻² SSDS1 3.0 × 10⁻²SS22A4 ND SS21B7 ND

[0405] TABLE 5 Round 1 No. Clones Selection phage taken screened Mab +ve % Mab + ve 1A — 94 2 2 1B — 94 0 0 2A 1A 48 6 13  2B 1A 65 5 8 2C 1A48 2 4 2D 1B 25 0 0

[0406]

1 36 645 base pairs nucleic acid double linear 1 GAATTCCGGA AAAAACAAAATTCCTGTAAA ACAAATTAAC TCCAGGAACT TAAAATTTAC 60 TCCAAGACAT TTCCCTCAAAACAAAGCAAA AAACCCCAGC AAAGATCGTT ACATCACAA 120 ACCAAACACA AAGACCAGCGGTCACAGGCA AGTTCCTCTA AGCTTCCATT CTGCTGACT 180 GTGGCTTCCA TTTAAAAGGAGTCTTTTAAT CAAGCCACTT TCACAGAATT TAAAACAAA 240 CAAACACATG TAAATTGCAAAATACAAAAA GGTAAATTTA TAAGTAAAAA TGACCAAAC 300 CACAAAACTG GAGTATTTCGAAGGTTGAGG GTTCAGTGGA GGGTGTAACA CGAAAGGAA 360 TTCACAACTG AAAGAAATCATTGCCGAGTT TCCTCCAGGC AGCACTGAAA TGAATGGAG 420 ACCTTCTCTC GAACATCTCACACGTTAAAA AAAATAAATA TTTAAGAGAT ACAAGGCTC 480 GATTGGTTTT CATATACATTGCACTTGAAG TTTAAGACCC AATACTTGCA AATTAGGTC 540 GGTATGGTTT ATGCCATTAAATGAATACAT TGTGCTCACC AATATCATTG ACTAGAAAC 600 CCACACGTTT AATGCAGTGCCATATGCAAT CTGTGACCGG AATTC 645 12 amino acids amino acid linear 2 GluPhe Arg Lys Lys Gln Asn Ser Cys Lys Thr Asn 1 5 10 45 amino acids aminoacid linear 3 Leu Gln Glu Leu Lys Ile Tyr Ser Lys Thr Phe Pro Ser LysGln Ser 1 5 10 15 Lys Lys Pro Gln Gln Arg Ser Leu His His Lys Thr LysHis Lys Asp 20 25 30 Gln Arg Ser Gln Ala Ser Ser Ser Lys Leu Pro Phe Cys35 40 45 5 amino acids amino acid linear 4 Leu Val Ala Ser Ile 1 5 50amino acids amino acid linear 5 Lys Glu Ser Phe Asn Gln Ala Thr Phe ThrGlu Phe Lys Thr Asn Gln 1 5 10 15 Thr His Val Asn Cys Lys Ile Gln LysGly Lys Phe Ile Ser Lys Asn 20 25 30 Asp Gln Thr His Lys Thr Gly Val PheArg Arg Leu Arg Val Gln Trp 35 40 45 Arg Val 50 19 amino acids aminoacid linear 6 His Glu Arg Asn Phe Thr Thr Glu Arg Asn His Cys Arg ValSer Ser 1 5 10 15 Arg Gln His 18 amino acids amino acid linear 7 Asn GluTrp Arg Thr Phe Ser Arg Thr Ser His Thr Leu Lys Lys Ile 1 5 10 15 AsnIle 32 amino acids amino acid linear 8 Glu Ile Gln Gly Ser Asp Trp PheSer Tyr Thr Leu His Leu Lys Phe 1 5 10 15 Lys Thr Gln Tyr Leu Gln IleArg Ser Gly Met Val Tyr Ala Ile Lys 20 25 30 8 amino acids amino acidlinear 9 Ile His Cys Ala His Gln Tyr His 1 5 6 amino acids amino acidlinear 10 Leu Glu Thr Pro His Val 1 5 7 amino acids amino acid linear 11Cys Ser Ala Ile Cys Asn Leu 1 5 83 amino acids amino acid linear 12 AsnSer Gly Lys Asn Lys Ile Pro Val Lys Gln Ile Asn Ser Arg Asn 1 5 10 15Leu Lys Phe Thr Pro Arg His Phe Pro Gln Asn Lys Ala Lys Asn Pro 20 25 30Ser Lys Asp Arg Tyr Ile Thr Lys Pro Asn Thr Lys Thr Ser Gly His 35 40 45Arg Gln Val Pro Leu Ser Phe His Ser Ala Asp Trp Trp Leu Pro Phe 50 55 60Lys Arg Ser Leu Leu Ile Lys Pro Leu Ser Gln Asn Leu Lys Gln Thr 65 70 7580 Lys His Met 9 amino acids amino acid linear 13 Ile Ala Lys Tyr LysLys Val Asn Leu 1 5 14 amino acids amino acid linear 14 Val Lys Met ThrLys Pro Thr Lys Leu Glu Tyr Phe Glu Gly 1 5 10 39 amino acids amino acidlinear 15 Gly Phe Ser Gly Gly Cys Asn Thr Lys Gly Thr Ser Gln Leu LysGlu 1 5 10 15 Ile Ile Ala Glu Phe Pro Pro Gly Ser Thr Glu Met Asn GlyGlu Pro 20 25 30 Ser Leu Glu His Leu Thr Arg 35 16 amino acids aminoacid linear 16 Ile Phe Lys Arg Tyr Lys Ala Gln Ile Gly Phe His Ile HisCys Thr 1 5 10 15 28 amino acids amino acid linear 17 Ser Leu Arg ProAsn Thr Cys Lys Leu Gly Leu Val Trp Phe Met Pro 1 5 10 15 Leu Asn GluTyr Ile Val Leu Thr Asn Ile Ile Asp 20 25 16 amino acids amino acidlinear 18 Lys His His Thr Phe Asn Ala Val Pro Tyr Ala Ile Cys Asp ArgAsn 1 5 10 15 8 amino acids amino acid linear 19 Ile Pro Glu Lys Thr LysPhe Leu 1 5 7 amino acids amino acid linear 20 Asn Lys Leu Thr Pro GlyThr 1 5 35 amino acids amino acid linear 21 Asn Leu Leu Gln Asp Ile SerLeu Lys Thr Lys Gln Lys Thr Pro Ala 1 5 10 15 Lys Ile Val Thr Ser GlnAsn Gln Thr Gln Arg Pro Ala Val Thr Gly 20 25 30 Lys Phe Leu 35 15 aminoacids amino acid linear 22 Ala Ser Ile Leu Leu Thr Gly Gly Phe His LeuLys Gly Val Phe 1 5 10 15 7 amino acids amino acid linear 23 Ser Ser HisPhe His Arg Ile 1 5 13 amino acids amino acid linear 24 Asn Lys Pro AsnThr Cys Lys Leu Gln Asn Thr Lys Arg 1 5 10 25 amino acids amino acidlinear 25 Pro Asn Pro Gln Asn Trp Ser Ile Ser Lys Val Glu Gly Ser ValGlu 1 5 10 15 Gly Val Thr Arg Lys Glu Leu His Asn 20 25 13 amino acidsamino acid linear 26 Lys Lys Ser Leu Pro Ser Phe Leu Gln Ala Ala Leu Lys1 5 10 33 amino acids amino acid linear 27 Met Glu Asn Leu Leu Ser AsnIle Ser His Val Lys Lys Asn Lys Tyr 1 5 10 15 Leu Arg Asp Thr Arg LeuArg Leu Val Phe Ile Tyr Ile Ala Leu Glu 20 25 30 Val 6 amino acids aminoacid linear 28 Asp Pro Ile Leu Ala Asn 1 5 7 amino acids amino acidlinear 29 Val Trp Tyr Gly Leu Cys His 1 5 28 amino acids amino acidlinear 30 Met Asn Thr Leu Cys Ser Pro Ile Ser Leu Thr Arg Asn Thr ThrArg 1 5 10 15 Leu Met Gln Cys His Met Gln Ser Val Thr Gly Ile 20 25 15amino acids amino acid linear 31 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser 1 5 10 15 20 amino acids amino acid linear 32 MetAsp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15Ser Glu Pro Cys 20 10 amino acids amino acid linear 33 Pro Met Pro HisAla Glu Gly Lys Ser Thr 1 5 10 14 amino acids amino acid linear 34 GlyAla Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 1 5 10 15 base pairsnucleic acid single linear 35 GACTCCTGGA GCCCG 15 15 base pairs nucleicacid single linear 36 CGCGGCCAGC GATGG 15

1. A method of labelling molecules, the method including providing in acommon medium: a label molecule; a ligand (“first marker ligand”) ableto bind a second member of a specific binding pair (sbp); a said secondsbp member; an enzyme able to catalyse binding of said label molecule toother molecules, said enzyme being associated with said first markerligand; causing or allowing binding of said first marker ligand to saidsecond sbp member; and causing or allowing binding of said labelmolecule to other molecules in the vicinity of said first marker ligandbound to said second sbp member.
 2. A method according to claim 1wherein said other molecules to which said label molecule binds includea sbp member (“first sbp member”) which binds said second sbp member. 3.A method according to claim 1 wherein said other molecules to which saidlabel molecule binds include a sbp member (“first sbp member”) whichbinds a molecule in the vicinity of said second sbp member.
 4. A methodaccording to claim 3 wherein said molecule in the vicinity of saidsecond sbp member is on a cell surface.
 5. A method according to claim 3wherein said molecule in the vicinity of said second sbp member iscomplexed with said second sbp member.
 6. A method according to claim 1wherein said second sbp member is displayed on the surface of a virusparticle.
 7. A method according to claim 1 wherein a diverse populationof said second sbp members is provided in the common medium, including asecond sbp member able to bind said marker ligand.
 8. A method accordingto claim 1 wherein the second sbp member is a peptide.
 9. A methodaccording to claim 2 wherein said first sbp member is displayed on thesurface of a virus particle.
 10. A method according to claim 2 wherein adiverse population of first sbp members is provided in the commonmedium, including a first sbp member able to bind said second sbpmember.
 11. A method according to claim 2 wherein said first sbp memberincludes an antibody antigen binding domain.
 12. A method according toclaim 11 wherein said first sbp member includes a scFv, Fab, Fv, dAb, Fdor diabody molecule.
 13. A method according to claim 1, wherein saidmarker ligand includes a cytokine or chemokine.
 14. A method accordingto claim 1 wherein said marker ligand includes an antibody antigenbinding domain.
 15. A method according to claim 2 wherein said markerligand includes a non-human antibody antigen binding domain.
 16. Amethod according to claim 15 wherein said first sbp member includes ahuman antibody antigen binding domain.
 17. A method according to claim1, wherein the label includes an activatible binding molecule.
 18. Amethod according to claim 17 wherein the activatible binding molecule istyramine.
 19. A method according to claim 1, wherein the enzyme ishydrogen peroxidase.
 20. A method according to claim 1, wherein theenzyme is conjugated to the marker ligand.
 21. A method according toclaim 20 wherein the enzyme is conjugated to the marker ligand via apeptidyl linkage.
 22. A method according to claim 21 wherein the enzymeis conjugated to the marker ligand via a peptide or polypeptide linker.23. A method according to claim 20 wherein the enzyme is conjugated tothe marker ligand via a member of a specific binding pair.
 24. A methodaccording to claim 20 wherein the enzyme is conjugated to the markerligand via a spacer.
 25. A method according to claim 24 wherein thespacer is a chemical linker, polymer, peptide, polypeptide, or rigidbead, phage molecule, or other particle.
 26. A method according to claim1, wherein following binding of said label molecule to other moleculesin the vicinity of said marker ligand bound to said second sbp member,labelled molecules are isolated and/or purified from the medium.
 27. Amethod according to claim 26 wherein said label is a member of aspecific binding pair.
 28. A method according to claim 27 whereinlabelled molecules are isolated and/or purified by means of binding ofsaid label to complementary sbp member.
 29. A method according to claim27 wherein said label is biotin.
 30. A method according to claim 29wherein biotinylated molecules are isolated and/or purified by means ofbinding of biotin to streptavidin.
 31. A method according to claim 2wherein a first sbp member labelled in accordance with the method isemployed as a second ligand (“second marker ligand”) in a furtherperformance of said method.
 32. A method according to claim 31 wherein afurther first sbp member labelled in accordance with said furtherperformance of said method is isolated and/or purified from the medium.33. A method according to claim 32 wherein said further first sbp memberis an agonist or antagonist of said first marker ligand.
 34. A methodaccording to claim 1, wherein the amount of labelling of in the vicinityof said first marker ligand bound to said second sbp member isdetermined.
 35. A method according to claim 26 wherein an isolatedand/or purified molecule is modified.
 36. A method according to claim 35wherein the isolated and/or purified molecule is a peptide orpolypeptide molecule and is modified by addition, insertion,substitution and/or deletion of one or more amino acids.
 37. A methodaccording to claim 35 wherein the isolated and/or purified molecule ismodified by linkage of another molecule.
 38. A method according to claim26 wherein an isolated and/or purified molecule or a derivative thereofis formulated into a composition which includes at least one additionalcomponent.
 39. A method according to claim 38 wherein said compositionincludes a pharmaceutically acceptable vehicle or carrier.
 40. A methodaccording to claim 1 wherein said second sbp member is displayed on thesurface of a virus particle and said particle is isolated and/orpurified following binding of said marker ligand to said second sbpmember and labelling of said particle and/or said displayed second sbpmember.
 41. A method according to claim 2 wherein said first sbp memberis displayed on the surface of a virus particle and said particle isisolated and/or purified following binding of said first sbp member tosaid second sbp member and labelling of said particle and/or saiddisplayed first sbp member.
 42. A method according to claim 3 whereinsaid first sbp member is displayed on the surface of a virus particleand said particle is isolated and/or purified following binding of saidfirst sbp member to said molecule in the vicinity of said second sbpmember and labelling of said particle and/or said displayed first sbpmember.
 43. A method according to claim 40 wherein said particle isisolated and/or purified by means of a member of a specific binding pairable to bind said labelled particle and/or displayed first or second sbpmember.
 44. A method according to claim 43 wherein following isolationand/or purification of said virus particle, nucleic acid is obtainedfrom said particle.
 45. A method according to claim 44 includingexpression from nucleic acid with the sequence of said nucleic acidobtained from said particle or a sequence modified by the addition,insertion, deletion and/or substitution of one or more nucleotides toproduce an encoded peptide or polypeptide.
 46. A method according toclaim 45 wherein the peptide or polypeptide produced by expression fromsaid nucleic acid is modified.
 47. A method according to claim 43wherein the peptide or polypeptide produced by expression from saidnucleic acid or a derivative thereof is formulated into a compositionincluding at least one additional component.
 48. A method according toclaim 47 wherein the composition includes a pharmaceutically acceptablevehicle or carrier.
 49. A method of producing a member of a specificbinding pair, the method including (i) producing by expression fromnucleic acid which has the sequence of nucleic acid obtained inaccordance with claim 44 an encoded peptide or polypeptide; or (ii)producing by expression from nucleic acid which has the sequence ofnucleic acid obtained in accordance with claim 44 modified by insertion,addition, deletion and/or substitution of one or more nucleotides anencoded peptide or polypeptide.
 50. A method according to claim 49wherein the encoded peptide or polypeptide is modified following itsproduction by expression.
 51. A method according to claim 49 wherein theencoded peptide or polypeptide or a derivative thereof is formulatedinto a composition including at least one additional component.
 52. Amethod according to claim 51 wherein the composition includes apharmaceutically acceptable vehicle or carrier.
 53. A method ofproducing nucleic acid encoding a member of a specific binding pair, themethod including (i) producing nucleic acid which encodes a member of aspecific binding pair which is encoded by the nucleic acid obtained inaccordance with claim 44; or (ii) producing nucleic acid which encodes aderivative, by way of addition, deletion, insertion and/or substitutionof one or more amino acids, of a member of a specific binding pair whichis encoded by the nucleic acid obtained in accordance with claim
 44. 54.A reaction medium containing: a label molecule; a ligand (“markerligand”) able to bind a second member of a specific binding pair member;a said second sbp member; and an enzyme able to catalyse binding of saidlabel molecule to other molecules, said enzyme being associated withsaid marker ligand.
 55. A reaction medium according to claim 54including a sbp member (“first sbp member”) able to bind said second sbpmember.
 56. A sbp member identified as having ability to bindcomplementary sbp member in accordance with a method according to claim26.
 57. A sbp member obtained in accordance with a method according toclaim
 49. 58. A sbp member according to claim 57 which includes anantibody antigen binding domain.
 59. A composition including a sbpmember obtained in accordance with a method according to claim 49 or aderivative thereof.
 60. A composition according to claim 59 whichincludes a pharmaceutically acceptable vehicle or carrier.
 61. Nucleicacid obtained in accordance with a method according to claim 53.